nutri-medicinal plants used in the management of hiv/aids...
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Nutri-medicinal plants used in the management of HIV/AIDS
opportunistic infections in western Uganda: documentation,
phytochemistry and bioactivity evaluation
SAVINA ASIIMWE
ROYAL INSTITUTE OF TECHNOLOGY MAKERERE UNIVERSITY
Doctoral Thesis
Stockholm 2015
Cover : Plectranthus amboinicus (Lour.) Spreng and one of its major chemical compound, carvacrol
(photo by Asiimwe Savina)
© Asiimwe Savina, 2015
ISBN 978-91-7595-649-7
ISSN 1654-1081
TRITA-CHE Report 2015:34
Printed by Universitesservice US-AB Stockholm
Savina Asiimwe, 2015: Nutri-medicinal plants used in the management of HIV/AIDS opportunistic
infections in western Uganda: documentation, phytochemistry and bioactivity evaluation. Chemistry,
KTH Chemical Science and Engineering, Royal Institute of Technology, SE-100 44 Stockholm, Sweden;
and Biological Sciences, College of Natural Sciences, Makerere University, P. O Box 7062, Kampala,
Uganda.
Abstract
As a result of the AIDS epidemic, many people are immunocompromised and opportunistic
infections are common. Medicinal plants constitute one of the fundaments of HIV treatment and
are commonly used in management of HIV – related ailments, and also to counteract the side
effects of antiretroviral therapy. This study documents and evaluates nutri-medicinal plants
traditionally used in the management of opportunistic infections associated with HIV/AIDS in
western Uganda. A six-stage process of documentation, evaluation and analysis of results was
conducted: (1) ethnobotanical studies leading to identification and documentation of medicinal
and nutritional plants most frequently used in the treatment of opportunistic infections of
HIV/AIDS (2) Collection of plant samples and preparation of the extracts of each of the selected
plants needed for bioactivity evaluation; (3) Phytochemical analysis of crude plant extracts
(qualitative and GC/MS analysis); (4) pharmacological evaluation of the crude plant extracts
(antimicrobial, antioxidant and mineral nutrient evaluation); (5) safety evaluation of the active
extracts using animal models, and (6) Statistical analysis of the results.
The study recorded 324 plant species distributed in 75 families, with potential to treat ailments
associated with immuno-compromised people living with HIV/AIDS in western Uganda. The
study revealed that folk medicine is still widely practiced. Fidelity level values indicated the
most preferred plant species for particular ailments. The high consensus values indicated that
there was high agreement in the use of plants for various ailments. The selected preferred plant
species were subjected to chemical screening to ascertain their pharmacological activities and
they could be prioritized for conservation. The study allows for identifying high value medicinal
plants indicating high potential for economic development.
Phytochemical screening of the aqueous and ethanol extracts of selected twenty plant species
revealed the presence of tannins, saponins, flavonoids, anthocyanins, coumarins and steroid
glycosides. Some of the major chemical compounds identified by gas chromatography mass
spectrometry of the essential oils include α- phellandrene, linalool, carvacrol, geraniol, β-
eudesmene, β-cubebene, α-caryophyllene, 1-8 cineole and caryophyllene oxide. The essential
oils of Plectranthus amboinicus, Erlangea tomentosa, Plunchea ovalis and Crassocephalum
vitellinum were highly active against Candida albicans and Cryptococcus neoformans. One of
the essential oil fractions of Crassocephalum vitellinum (1.56 mg/ml) was highly active against
Cryptococcus neoformans. Antioxidant activities of the plant species were also tested. The
antioxidant activity of Pseudarthria hookeri (43.68%) and the ferric reducing power of
Symphytum officinale (10.48 Mm/L) were the highest values. The ability of the plant extracts to
scavenge free radicals may partly justify the traditional use of these plants to boost immunity in
HIV/AIDS patients. Mineral nutrient analysis revealed high amounts of iron in Plectranthus
amboinicus (5.8 mg/kg dry weight), zinc in Pseudarthria hookeri (6.9 mg/kg dry weight) and
selenium in Plunchea ovalis (1.14 mg/kg dry weight). These elements are essential in
maintenance of the immune system. Hematological analysis of the aqueous extract of
Plectranthus amboinicus showed that the plant has immunostimulating properties by increasing
the number of lymphocytes in the test animals. Further ethnopharmacological studies are needed
for the documented plants particularly the most active ones.
Key words: Ethnobotanical study, Medicinal plants, HIV, AIDS, opportunistic infections,
bacteria, fungi, GC-MS, phytochemistry, antioxidant, histopathology, biochemistry, hematology,
western Uganda.
List of Publications and Manuscripts
This thesis is based on the following papers and manuscripts which will be referred to in the text
by Roman numerals as follows:
I. Ethnobotanical study of nutri-medicinal plants used for the management of HIV/AIDS
opportunistic ailments among the local communities of western Uganda. Savina
Asiimwe, Maud Kamatenesi-Mugisha, Agnes Namutebi, Anna Karin Borg-Karlson,
Peace Musiimenta.
Journal of Ethnopharmacology 150(2013)639–648
II. Documentation and consensus of indigenous knowledge on medicinal plants used by the
local communities of western Uganda. Savina Asiimwe, Agnes Namutebi, Anna Karin
Borg-Karlson, Maud Kamatenesi Mugisha and Hannington Oryem-Origa.
Journal of Natural Product and Plant Resources, 2014,4 (1):34-42
III. Ethnobotanical study of indigenous knowledge on medicinal and nutritious plants used to
manage opportunistic infections associated with HIV/AIDS in western Uganda. Maud
Kamatenesi Mugisha, Savina Asiimwe, Agnes Namutebi, Anna Karin Borg- Karlson, and
Esezah Kyomugisha Kakudidi.
Journal of Ethnopharmacology 155(2014)194–202
IV. Chemical composition and Toxicological evaluation of the aqueous leaf extracts of
Plectranthus amboinicus Lour. Spreng. Savina Asiimwe., Anna Karin Borg- Karlson.,
Muhammad Azeem., Kamatenesi Maud Mugisha., Agnes Namutebi and Ndukui James
Gakunga.
International Journal of Pharmaceutical Science Invention February 2014, 4 (2):19-27]
V. Chemical composition and antimicrobial evaluation of the essential oil and fractions
obtained from Plectranthus amboinicus (Lour.) Spreng traditionally used in the
management of HIV/AIDS opportunistic infections. Savina Asiimwe, Anna Karin Borg
Karlson, Muhammad Azeem, Abier Hamed Sofrata, Robert Byamukama, Maud
Kamatenesi- Mugisha and Agnes Namutebi.
Manuscript for Phytochemistry
VI . Phytochemical screening, antioxidant activities and mineral composition of nutri-
medicinal plants used in the management of opportunistic ailments in HIV/AIDS
patients. Savina Asiimwe, Agnes Namutebi, Maud Kamatenesi Mugisha, and Anna Karin
Borg- Karlson.
Manuscript for Nutrition
Abbreviations
AIDS Acquired Immune Deficiency Syndrome
ANOVA Analysis of Variance
ART Antiretroviral therapy
CD4 Cluster of differentiation 4
DPPH 1,1- Diphenyl -2 - picrylhydrazyl
DMSO Dimethyl sulphoxide
FRAP Ferric reducing antioxidant power
GC-MS Gas Chromatography Mass Spectrometry
HAART Highly active antiretroviral therapy
HS-SPME Head space solid phase microextraction
HIV Human Immunodeficiency Virus
IK Indigenous Knowledge
LD50 Dose that causes mortality in 50 % in organisms tested
MBC/MFC Minimum bacterial concentration and minimum fungicidal concentration
MPLC Medium pressure liquid chromatography
UNAIDS United Nations Programme on HIV/AIDS
THETA Traditional and Modern Health Practitioners Together Against AIDS
TBAs Traditional Birth Attendants
TM Traditional Medicine
TK Traditional Knowledge
WHO World Health Organization
OECD Organization for Economic Corporation Development
PLWHA People living with HIV/AIDS
UNGASS United Nations General Assembly Special Session
Thesis outline
This thesis is composed of chapters that have been published in various peer accredited reviewed
journals or are in preparation. Chapter one gives an overview of the problem of HIV/AIDS in
Uganda and a worldwide perspective. It also gives a general overview of how nutri-medicinal
plants are traditionally used in the management opportunistic infections of HIV/AIDS;
objectives, research questions, problem statement and significance of the study. Chapter two
describes the methods used in data collection during the study which include ethnobotanical
survey of plants used, investigations of the phytochemical constituents and antimicrobial
activities of plant extracts. The isolation of the active compounds in the plant extracts through
bioassay – guided fractionation is also discussed therein. The antioxidant activities and toxicity
evaluation of the plant extracts is presented in chapter three. Chapter four presents results and
general discussion as well as published articles and manuscripts which are in preparation.
Chapter 5 outlines conclusions and recommendations from the study in the perspective of
contributing knowledge to this pertinent subject.
Dedication
I dedicate this piece of work to my beloved daughter Asiimwe Josephine Byarugaba for her
endurance and support during the times I would be away from home. To my husband Dominic
Byarugaba, my sons Derrick Taremwa and Davis Twiine who always support my endeavours.
To my friends for helping me achieve such an honored accomplishment and to many individuals
that are continuously striving for an integrated health care delivery system in Uganda.
Table of Contents
Abstract
List of Publications and manuscripts
Abbreviations
Thesis outline
1. Introduction ............................................................................................................................................... 1
1.1 The origin of HIV and the AIDS pandemic .......................................................................................... 1
1.2 Traditional medicine in the management of opportunistic infections of HIV/ AIDS ......................... 3
1.3 Major groups of antimicrobial compounds from plants ....................................................................... 4
1.4 Common opportunistic bacterial and fungal organisms in HIV-infected patients ............................... 6
1.5 Antioxidants in the pathogenesis of HIV/AIDS: A healthy immune system ....................................... 7
1.6 Safety evaluation of nutri-medicinal plants: the need for toxicity testing of herbal extracts ............... 9
1.7 Problem Statement ............................................................................................................................. 10
1.8 Significance of the study .................................................................................................................... 11
1.9 Research objectives ............................................................................................................................ 12
1.9.1 Main objective ............................................................................................................................. 12
1.9.2 Specific objectives ....................................................................................................................... 12
1.9.3 Research questions ....................................................................................................................... 12
2. Materials and methods ............................................................................................................................ 13
2.1 Description of study sites ................................................................................................................... 13
2.2 Ethnobotanical field survey ............................................................................................................... 15
2.3 Selection of plants and extract preparation for bioactivity evaluation ............................................... 16
2.4 Qualitative phytochemical analysis of crude extracts ........................................................................ 17
2.4.1 The use of water and ethanol in phytochemistry ........................................................................ 17
2.4.2 Chemical qualitative analysis of plant extracts ......................................................................... 18
2.5 Collection of headspace volatiles, solid phase microextraction ....................................................... 20
2.6 Isolation of essential oil .................................................................................................................. 20
2.7 Fractionation of essential oil using medium pressure chromatography ........................................... 21
2.8 Chemical analyses of essential oil ................................................................................................... 22
2.9 Microorganisms and preparation of sample extracts used for antimicrobial assay .......................... 23
2.10 Antimicrobial assay of the crude extracts ...................................................................................... 23
2.10.1 Determination of Minimum inhibitory concentrations .......................................................... 24
2.10.2 Determination of MBCs and MFCs ...................................................................................... 24
2.10.3 Growth curve studies using Bioscreen method ...................................................................... 25
3. Analysis of plant extracts for nutritive values........................................................................................ 26
3.1 Evaluation of total antioxidant capacity by FRAP method .............................................................. 26
3.2 Evaluation of total antioxidant capacity by DPPH .......................................................................... 26
3.3 Mineral nutrient analysis using atomic absorption spectrometry ..................................................... 27
3.4 Safety evaluation of selected plant extracts ..................................................................................... 27
3.4.1 Acute toxicity ........................................................................................................................... 27
3.4.2 Sub acute toxicity ..................................................................................................................... 28
3.5 Statistical analysis of data ............................................................................................................... 29
3.5.1 Quantitative analysis of ethnobotanical data............................................................................. 29
3.5.2 Analysis of toxicity results ........................................................................................................ 29
4. Results and discussion ............................................................................................................................ 30
4.1 Ethnobotanical studies - documentation of nutri-medicinal plants .................................................. 30
4.2 Phytochemistry, antimicrobial and antioxidant activities of nutri-medicinal plants ........................ 33
4.2.1 Chromatographic essential oil analysis ...................................................................................... 33
4.2.2 Major groups of volatile compounds identified in the essential oils .......................................... 34
4.2.3 Antibacterial and antifungal testing of plant extracts................................................................ 36
4.2.4 Growth curve studies using Bioscreen method ......................................................................... 39
4.2.5 Antioxidant capacity and mineral nutrient composition ........................................................... 41
4.3 Safety evaluation of nutri-medicinal plants ....................................................................................... 42
4.3.1 Acute toxicity study ..................................................................................................................... 42
4.3.2 Sub acute toxicity studies ............................................................................................................. 44
5. Conclusions ............................................................................................................................................. 46
6. Acknowledgements ................................................................................................................................ 49
7. References ............................................................................................................................................... 51
Appendix A: Author’s contribution ....................................................................................................... 61
Appendix B: Structures of major compounds identified by GC/MS ................................................... .62
Appendix C: Preliminary antimicrobial screening results ..................................................................... 63
Appendix D: Antimicrobial screening of the four active plant extracts………………………...……..66
Appendix E. Toxicity signs observed during toxicity studies…………….………………..…….…....69
Appendix F. Chromatograms of essential oils of some active plant species…….…………….……....70
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1. Introduction
The work undertaken in this thesis is part of a broader project aimed at integration of nutri-
medicinal plants as therapeutics in health care delivery in the western region of Uganda. The
main goal of the project is to identify, upgrade and integrate nutri-medicinal plants used in the
management of opportunistic ailments including HIV/AIDS and contribute to improved health in
Uganda. The project involves documenting and validating of indigenous plants that are used for
medicinal as well as nutritional values, by conducting ethnopharmacological and nutritional
screening of selected plants. The project also aims at initiating propagation and domestication of
key, rare and endangered nutri-medicinal plants for wide scale usage in communities for targeted
ailments in immuno-compromised groups of people and to train local communities on techniques
of multiplication of indigenous medicinal plants used in the management of HIV/AIDS and other
opportunistic infections. Cultivation and domestication are central in this project so as to add
value to African nutri-medicinal plants for easy accessibility to the wider community. An
assortment of key plant species used in the management of HIV/AIDS clustered as backyard
orchard plants will go a long way in the conservation efforts of these key species.
1.1 The origin of HIV and the AIDS pandemic
Acquired immunodeficiency syndrome (AIDS) is a chronic and potentially fatal disease of the
immune system caused by the human immunodeficiency virus (HIV). The virus attacks a
specific type of white blood cells known as T-lymphocytes which are critical to the normal
functioning of the human immune system that defends the body against all types of illnesses.
These cells are measured in the blood as CD4 count, and the lower the CD4 count, the weaker
the immune system. Healthy people have between 600-1200 cells/mm3 of blood. When CD4
cells count is less than 200 cells/mm3, this is considered an advanced stage of HIV called AIDS.
Therefore, CD4 cells act to control and direct the immune response and are used to monitor the
progress of HIV infection. The consequences of a weak immune system in HIV/AIDS include
the manifestations of opportunistic infections of which skin diseases form a large portion
(Filiberto et al., 2011). Opportunistic infections involve multiple systems of the body such as
immune, gastrointestinal, genitourinary, endocrine, dermatological and nervous system.
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HIV belongs to the lentivirus, subfamily of the retrovidae and is a single-stranded RNA virus
(Armbruster, 2008). AIDS in humans is caused by two lentiviruses, HIV-1 and HIV-2, identified
in 1983 and 1986 respectively. HIV-1 has long been suspected to be of chimpanzee origin (Gao
et al., 1999). Both HIVs are a result of multiple cross species transmissions of simian
immunodeficiency virus (SVI) naturally infecting African primates (Sharp and Hahn, 2011;
Sharp et al., 2005). However, one transmission event, involving SIVcpz from chimpanzees in
southeastern Cameroon gave rise to HIV-1 group M; the principle cause of the AIDS pandemic,
which has infected millions of people worldwide and is found in virtually every country on the
globe (Reeves and Doms, 2002; Sharp and Hahn, 2011). In humans, HIV can be transmitted
through infected blood, semen, vaginal secretions, breast milk, and during pregnancy or delivery
of a new born.
The first AIDS case was diagnosed in 1981 in the United States, when cases of rare diseases
caused by unknown virus among homosexual men were reported. Such cases include pneumonia
caused by pneumocyctis carinii and Karposi sarcoma, a rare skin cancer caused by a virus
(Gottlieb et al., 1981). Since then, the disease has spread to epidemic proportions around the
world. HIV/AIDS affects mankind globally, but the highest prevalence is in Sub-Saharan Africa
(with 68% of all cases of HIV/AIDS and a prevalence above 15% in some regions), and in some
areas of South-East and Central Asia. In 2013, about 35 million people globally were living with
the HIV infection, 2.1 million people became newly infected with HIV, while 1.5 million people
died from AIDS- related illnesses (UNAIDS, 2014). However, treatment is not available globally
and UNAIDS still estimates that there are currently 5000 AIDS – related deaths worldwide per
day (UNAIDS, 2013).
In Uganda, the first case was identified in 1982 along the shores of Lake Victoria (Mugerwa et
al., 1996). Consequently, the epidemic spread very fast to all parts of the country initially
concentrating in urban and semi-urban centers (Mugerwa et al., 1996). By the end of 1992, the
national prevalence rate was estimated at 18.3% with some centers registering rates above 30%
(Mugerwa et al., 1996). This was followed by a steady decline in prevalence rates from the mid
1990s to 2000 to around 6% attributable to favourable prevention policies. Uganda’s HIV sero-
prevalence rate of 7.3% among adults and 0.7% among children is among the world’s highest
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(UNAIDS, 2013), with an estimated 1.3 million people living with HIV (Lamorde et al., 2010).
About 43% of new infections occur among married people in monogamous heterosexual
relationships. The HIV pandemic is currently the most socio-economic challenge that faces the
most economic productive sectors of society – the young and women. The western region of
Uganda has the most marked variability and antenatal HIV prevalence, with Mbarara district
recording the highest HIV prevalence rate of 13.7% in the region (UNGASS, 2010). Poverty,
underdevelopment and illiteracy contribute to the spread of HIV in the developing world, yet
HIV/AIDS is also observed to aggravate the poverty situation thus hindering development
efforts. This calls for a fast mechanism of intervention by applying all forms of remedy, to
combat the alarming rates.
1.2 Traditional medicine in the management of opportunistic infections of HIV/AIDS
Traditional medicine is the sum total of the knowledge, skills, and practices based on the
theories, beliefs, and experiences indigenous to different cultures, whether explicable or not,
used in the maintenance of health as well as in the prevention, diagnosis, improvement or
treatment of physical and mental illness (WHO, 2002b). Traditional knowledge (TK) of
medicinal plants and their use by indigenous cultures is not only useful for conservation of
cultural traditions and biodiversity, but also for community healthcare and drug development for
present and future generations (Pei, 2001). In Africa and Asia, 80% of the population still uses
traditional remedies rather than modern medicine for primary health care (WHO, 2002a).
The pharmacological treatment of disease began long ago with the use of herbs, and methods of
folk healing throughout the world used herbs as part of their tradition (Schulz et al., 2001).
Traditional medicine has demonstrated its contribution to health through reduction of excessive
mortality, morbidity and disability due to diseases such as HIV/AIDS, malaria, tuberculosis,
diabetes, sickle cell anemia and mental disorders. The devastating effect of HIV/AIDS pandemic
coupled with the severe shortage of health personnel has compelled patients to develop coping
mechanisms by adopting alternative sources of primary health care. One of the sources is the use
of herbal therapies because they are easily accessible and affordable especially in rural settings.
In Africa, herbal medicines are used as primary treatment for HIV/AIDS and related infections
including insomnia, nausea, dermatological disorders and weakness (Hodgson and Rachanis,
4
2002; Mills et al., 2005). Studies have shown that majority of people living with HIV and
receiving antiretroviral drugs use herbal medicine after HIV diagnosis mainly to offset the side
effects from antiretroviral treatment (Kazhila and Marius, 2010) and to treat opportunistic
infections associated with the disease (Kisangau et al., 2007; Langlois-Klassen et al., 2007;
Peltzer et al., 2008). This is because patients on highly active antiretroviral therapy (HAART)
suffer side effects like poor appetite and nausea due to interference with absorption and
utilization of nutrients (Ridder, 2003).
However, research shows that traditional knowledge (TK) and plant diversity are currently being
lost at an accelerated rate by such forces as climate change, habitat destruction and globalization
(Thomas et al., 2009). Due to continuous destruction of habitats, numerous undiscovered species
are lost from the wild unrecorded. TK systems are also fading away along with cultures they
once kept alive. Hence, the aim of this study was to document and validate the medicinal plants
that double as medicine and food supplements in health care delivery especially in the
management of opportunistic infections associated with HIV/AIDS in western Uganda. It is
therefore important to document the uses of medicinal plants because such information can help
preserve the medicinal plant resources and the knowledge associated with them. The work
presented in this thesis contributes to the understanding of herbal medicine in managing
opportunistic ailments associated with HIV/AIDS in western Uganda.
1.3 Major groups of antimicrobial compounds from plants
Infectious diseases caused by bacteria, fungi, insects and arthropods are major threats to public
health despite tremendous progress that has been made in the medicinal sciences (Cos et al.,
2006). Microbial infections are the most frequent opportunistic diseases occurring during
HIV/AIDS which affect many people in Africa (Coulidiati et al., 2011). The search for
substances with antimicrobial activity from plants have been considered interesting by
researchers since they are frequently used in traditional remedies for many infectious diseases. A
number of modern drugs have been isolated from natural sources as remedies for human and
animal diseases as they contain phytochemicals of therapeutic value (de Melol et al., 2011;
Edeoga et al., 2005; Raginee et al., 2013). Different plant parts (root, leaves, stem bark, flowers
and seeds) are traditionally used to cure various ailments and diseases such as malaria, cough,
5
skin infections, asthma, diarrhoea and cancer among others. The plant parts produce a wide
range of chemical compounds such as alkaloids, saponins, tannins, flavonoids, terpenes, phenols
and essential oils that can be used to treat various chronic and infectious diseases (Cowan, 1999;
de Melol et al., 2011; Murakami et al., 2000; Periyanayagam et al., 2013; Reichling, 2010;
Sukirtha and Growther, 2012). For instance, tannins have been reported to hinder development of
microorganisms by their ability to precipitate and inactivate microbial adhesions enzymes and
cell envelops proteins (Cowan, 1999). Steroidal saponins have been reported to exhibit
antimicrobial activities against such organisms as Cryptococcus neoformans and Candida
species (Abbasołu and Türköz, 1995). Analysis of essential oils (mixtures of chemical
compounds obtained from aromatic herbs, flowers and fruits) shows that they contain different
compounds of which terpenoids in form of mono-, sesqui- and hemiterpenes are the most
abundant (Loza-Tavera, 1999; Trombetta et al., 2005). This abundance is equally related to the
soil type and general ecological and climatic setting of their location.
According to research, it takes years for a new drug to get through the research and development
pipeline to manufacture and the cost is quite enormous. In addition to this, drug resistance in part
caused by the misuse of medications, has rendered several antibiotics and other life-saving drugs
useless. Both these trends mean that scientists and pharmaceutical companies are urgently
looking for new drug sources and are increasingly turning their eyes to traditional medicine.
Research shows that almost a quarter of all modern medicines are derived from natural products,
many of which were first used in traditional remedies centuries ago. This study focused on
natural plant products as a useful source of antimicrobial molecules, active in particular, on
bacteria and fungi, as the main causes of infection in HIV/AIDS people. Studies exploiting the
mechanism of action and the structure- activity aspects of these natural compounds may provide
both additional antimicrobial leads and drugs, and also significant insight into potential
possibilities to overcome the antimicrobial resistance. Hence, the aim of this study was to
provide insights regarding the possibilities of the most important natural antimicrobial
compounds derived from plant sources containing a wide variety of secondary metabolites,
which are useful as alternative strategies to control microbial infections in HIV/AIDS patients.
As a result, many herbalists stand to set up more therapy centers sometimes to receive greater
numbers from time to time.
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1.4 Common opportunistic bacterial and fungal organisms in HIV-infected patients
Opportunistic infections (OIs) are infections that take advantage of a weak immune system.
When the immune system is weakened by HIV disease, bacteria and fungi can get out of control
and cause a wide range of illnesses resulting in mild to life threatening illnesses such as
diarrhoea, bacterial meningitis, respiratory infections, fungal infections of the skin, headache,
weight loss, night sweats, fever, cryptococcal meningitis, herpes zoster, candidiasis (fungal
infection of the oral cavity, throat and vagina), tuberculosis (a bacterial infection that attacks the
lungs), Pneumocystis carinii pneumonia and Mycobacterium avium complex which causes
recurring fevers (CDC, 2013; Murray and Pizzorno, 1999; Vermani and Garg, 2002). Patients
with HIV infection are particularly prone to infections with organisms such as Candida albicans,
Cryptococcus neoformans, Mycobacterium tuberculosis, Pseudomonas aeruginosa,
Streptococcus pneumoniae, Klebsiella pneumoniae and Staphylococcus aureus (methicillin-
susceptible and -resistant strains) are some of the most common pathogens of immuno-
compromised individuals (Zgoda and Porter, 2001). Klebsiella pneumonia is a causative
organism of pneumonia. Pseudomonas aeruginosa are residents of the intestinal tract and in
moist areas of the human skin. They cause a variety of systemic infections notably finger, toe
gaps, thigh and armpits in patients with cancer, immunocompromised patients with AIDS,
respiratory tract infections (Pseudomonas pneumonia), urinary tract, eye infections and wounds.
P. aeruginosa is notorious for its resistance to several antibiotics (Cooper et al., 2011; Philip et
al., 2009). Candida albicans is a causal agent of opportunistic oral and genital infections in
humans. S. aureus causes eye infections and is frequently found in the human respiratory tract
and on the skin.
Acute bacterial and fungal infections have been important causes of morbidity and mortality
especially among patients immunosuppressed by HIV/AIDS. Pneumococcal pneumoniae is
recognized as one of the leading early HIV-related problem and susceptibility increases with
progressive immunosuppression (Schuchat et al., 1991). According to world health organization,
increasing access to antiretroviral therapy (ART) has transformed the prognosis of HIV infected
patients. However, treatment coverage still remains low. As a result, many patients continue to
die of HIV-related opportunistic infections (OIs). Cryptococcal meningitis is one of the most
7
important opportunistic infection that causes more than 500,000 deaths in sub Saharan Africa
(Park et al., 2009).
1.5 Nutrition and antioxidants in the pathogenesis of HIV/AIDS: A healthy immune system
HIV has been considered the main cause in the progression of AIDS and it weakens the
defensive role of the human immune system (Anthony and Ashok, 2011). Therefore, the human
body requires a balanced diet that provides nutrients, minerals and vitamins for a functional and
effective immune system. The major immunity boosting components in herbs, fruits and
vegetables include flavonoids, phenols and carotenoids which are antioxidants that protect cells
from oxidative damage. Flavonoids have been reported to stimulate the immune system, reduce
platelet aggregation, reduce blood pressure and have antioxidant and antimicrobial effects
(Craig, 1999). Carotenoids boost the immune system to fight bacteria by increasing the number
of white blood cells.
Antioxidants are molecules that can delay or prevent an oxidative reaction catalyzed by free
radicals. It has been observed that perturbations in the antioxidant defense systems and
consequently redox imbalance are present in many tissues of HIV infected patients (Pham-Huy
et al., 2008). Free radicals and antioxidants are produced either from normal cell metabolism or
from external sources such as cigarette smoking, pollution, radiation or medication, and they
play a dual role as both toxic and beneficial compounds (Phum-Huy and Pham, 2008; Saikat et
al., 2010). However, accumulation of free radicals in the body results into a phenomenon called
oxidative stress, which leads to chronic and degenerative illnesses such as autoimmune disorders,
cancer, cardiovascular and neurodegenerative diseases (Pham-Huy et al., 2008). Hence,
antioxidants act as free radical scavengers by preventing and repairing damages caused by
reactive oxygen species, thereby enhancing the immune defense system and lowering the risk of
degenerative diseases. Some of the vital phytochemical metabolites include poly-phenols,
quinones, flavonoids, catechins, coumarins, terpenoids and in addition to the smaller molecules
like ascorbic acid (Vitamin C) and α-tocopherol (Vitamin E). Fruits, vegetables and medicinal
herbs are the richest sources of antioxidant compounds (Sies et al., 1992).
Nutrition in terms of antioxidants and trace minerals is integral to the care of all patients infected
with human immunodeficiency virus (HIV) by supporting their natural immunity (Nkengfack et
8
al., 2012). Minerals like selenium, zinc, iron, copper, manganese etc are well known antioxidants
(Shirwaikar et al., 2004) and are necessary to strengthen the body's own antioxidant protection /
immune system. Many studies link low levels of serum micronutrients with worsened HIV
disease status and mortality, for instance, low selenium (Kupka et al., 2004), low zinc (Lai et al.,
2001). Research shows that combining selenium with antiretroviral therapy (ART) suppresses
the progression of viral load in HIV and boosts numbers of immune cells (Stephen, 2008b). It
has also been reported that zinc affects a large number of HIV individuals (Jones et al., 2006)
and is a cofactor in more than 300 enzymes that influence different organ functions having a
secondary effect on the human immune system (Lothar and Philip, 2000). For instance, daily
intake of zinc reduces diarrhoea and increases weight gain.
Plants used traditionally as sources of food and medicine constitute a potentially useful resource
for new and safe drugs for the treatment of opportunistic infections. According to World Health
Organization (WHO, 2003), nutritional support is an integral part of a comprehensive response
to HIV/AIDS, helping to maintain the immune system and sustain healthy levels of physical
activity. It has been reported that HIV infection can jeopardize the body’s efficiency to utilize
food nutrients and can lead to malnutrition; which in turn can contribute to increased
immunocompromised state (Hecker and Kotler, 1990). In this way, proper nutrition enables HIV
patients to take medication, manage side effects from antiretroviral drugs and maintain adequate
nourishment by restoring intestinal function and weight gain (Guarino et al., 2002; Tinnerello,
1998). The beneficial effects of plant materials result from the combinations of secondary
products (phytochemicals) present in the plant. These phytochemicals can be used in treating
multiple illnesses such as malaria, HIV/AIDS and cancer among others. Studies have suggested
that the mechanisms responsible for progression of AIDS could be reversed through
administration of antioxidant reducing agents. Hence, this study was set out to determine the
nutritive values of selected plants in terms of total antioxidant capacity and mineral nutrient
composition.
9
1.6 Safety evaluation of nutri-medicinal plants: the need for toxicity testing of herbal
extracts
Toxicity studies show how potent a chemical is by measuring the concentration that will affect
the test organisms, thereby assessing the potential health risk in humans caused by intrinsic
adverse effects of chemical compounds present in plant extracts. Plants have been used for
medicinal purposes for centuries and are usually promoted as being ‘natural’ and ‘safe
alternatives’ to conventional medicines, but many contain useful as well as toxic constituents
(Adewunmi and Ojewole, 2004). Traditionally, people think that medicinal herbs being natural
are safe and free from undesirable effects failing to recognize that herbs are composed of
chemical compounds some of which may be toxic. For instance, Amaranthus species contain
high levels of oxalic acid, which has the ability to bind metals like calcium and magnesium
thereby interfering with their metabolism (Soetan and Aiyelaagbe, 2009). Although more than
80% of people today depend on herbal medication as a component of their primary health care
according to world health organization, there is still great concern about the safety and efficacy
of herbal use. Care must be taken not to consume harmful plants or high doses of plant extracts
that could have deleterious effects on vital body organs either in short or long term. Apart from
efficacy, safety of herbal medicines is of paramount importance as there is limited scientific
evidence to establish the safety and efficacy of most herbal products used in traditional medicine.
Although there are no systemic side-effects reported for humans in the literature (Jon and Ted,
2008), many different side effects have been reported owing to active ingredients, contaminants
or interactions with drugs (Stephen, 2008a). Herbal medicines have stood a test of time for their
safety, efficacy, cultural acceptability and lesser side effects. The chemical constituents present
in them are a part of the physiological functions of living flora and hence they are believed to
have better compatibility with the human body. This scenario withstanding, it is important to
determine phyto-extract toxicological effects for safety.
Biochemical tests have immense benefits in the diagnosis and monitoring of liver diseases
(Vasudevan and Sreekumari., 2007). The liver being the major organ responsible for metabolism
of toxic substances entering the body, its functions can be altered by liver injury following acute
or chronic exposure of toxicants (Alisi and Onyeze, 2008). Damage to the liver is associated with
cellular necrosis and increase in serum levels of biochemical parameters like Alanine
10
aminotransferase, bilirubin and aspartate aminotransferase (Wolf, 1999). Evaluation of
hematological parameters can also be used to determine the extent of deleterious effect of
xenobiotics (such as saponins, alkaloids and tannins) on the blood of an animal. Such analysis is
relevant to risk evaluation as changes in the hematological system have higher predictive value
for humans when the data are translated from animal studies. The adverse effects may manifest
in form of alterations in levels of biomolecules such as enzymes and metabolic products, normal
functioning and histomorphology of the organs (Ashafa et al., 2009). Hence, there is need to
study the effects of these plants which have potential therapeutic benefits to ascertain their safety
in animals using biochemical and haematogical indices.
Several studies have indicated the possibility that using plant extracts in high doses could lead to
toxic injury to the kidneys which interfere with renal tubular functions and induce acute renal
failure (Bwititi et al., 2000; Ijeh and Agbo, 2006). Research shows that most of the existing data
on traditional medicine in Africa deal only with medicinal plants and their uses, ignoring
chemical and pharmacological studies. The administration of herbal preparations without any
standard dosage coupled with inadequate scientific studies of their safety has raised concerns on
their toxicity. Hence, this study was done to evaluate the toxicological effects of five selected
plants used in traditional medicine to manage opportunistic ailments associated with HIV/AIDS
and other immunocompromised groups of people, such as the children and pregnant mothers.
1.7 Problem statement
The last two decades have witnessed dramatic rise in the incidence of life threatening
opportunistic infections due to the growth in the immunocompromised population especially
HIV/AIDS patients. The challenge has been to develop effective strategies for the management
of these infections in HIV patients. Despite the introduction of antiretroviral therapy (ART) to
arrest progression of immunodeficiency, less than 10% of HIV infected people have access to
any therapy for HIV infection. The development of drug resistance, lack of curative effect, high
cost, toxicity and unavailability are some of the shortcomings associated with the use of ART.
These shortcomings have resulted in increased efforts to search for better alternatives from
natural products. The use of herbal remedies is widespread among those living with HIV in
Uganda and more than 80% of Ugandans depend on traditional medicine for primary health care
11
needs. Even with the few plants documented through ethnobotanical surveys, little work has
been done on the screening of these plants to prove the validity of the claimed medicinal uses as
described by traditional healers. Coupled to this shortcoming is the secrecy traditional medicine
practitioners attach to their knowledge of herbal medicine.
Plants have not received their share of research especially validation in terms of safety, much as
organizations like THETA have promoted them in management of HIV/AIDS in Uganda. Above
all, health issues are continually challenging as pathogens evolve and the environment changes
from time to time; a situation that calls for constant research and testing of remedies. Plants are
widely used but their effects are still largely unknown in science studies, and nutrition which is a
key ingredient in medical treatment is ignored in most scientific research. The pharmacological
activities and toxicity profiles of most herbal drugs is unknown, and hence, the need to develop
cheaper, safer and effective antimicrobial drugs through research.
1.8 Significance of the study
Documentation of plant species with active compounds against HIV/AIDS opportunistic
infections helps to preserve or keep important indigenous knowledge safe. The key to medicinal
plants research revolves around the detection, isolation and characterization of active compounds
as therapeutic agents. Scientific validation of herbal medicines provides basic understanding of a
plant’s efficacy and this may lend further support to the widespread use of TM in health care
systems, provided toxicological investigations are carried out. Several herbs have been attributed
to improve immune response and could reduce symptoms of HIV/AIDS; however, this needs
scientific validation in terms of safety and efficacy. Information on safety and toxic levels of the
plants can be a starting point for the formulation of dosage levels of the herbal remedies.
Bioactivity analysis may lead to the identification and isolation of novel chemical compounds
and may confirm which plants are useful as nutraceutical and pharmaceutical products in the
management of HIV/AIDS related infections. With increasing antibiotic resistance, it is
important to find effective treatments for microbial infections. However, routine testing for
chemical composition, biological activity and toxicity of plant derived natural products are all
critical attributes to be considered for a complete understanding of the effect of medicinal plants
on human health care. The rationale of the study was to address the health burden in terms of
12
both medicine and nutrition intervention. This was done stemming from the first principles of
use which is indigenous knowledge information. This is why this study was set out to document
and validate the herbal and nutritive plants; and their practices in the management of HIV/
AIDS- related infections. Efforts geared towards improving the quality of herbal medicines will
in turn improve the quality of health, nutritional status and economic empowerment of the people
involved in the conservation, collection, preparation and administration of these plant products.
Hence the value of this research lies not only in promoting the value of nutri-medicinal plants in
the context of HIV/AIDS epidemic, but also in the need for improved primary health care. This
research will also create new knowledge in the area of pharmacology and nutrition.
1.9 Objectives
1.9.1 Main objective
The overall objective of the study was to document and validate the efficacy of nutri-medicinal
plants used to manage HIV/AIDS opportunistic ailments.
1.9.2 Specific objectives
The specific objectives of the study were to:
1. Identify and document plant species with nutritional and medicinal properties.
2. Determine the antioxidant and antimicrobial properties of the selected plant species.
3. Identify the bioactive molecules in the most active plant extracts.
4. Determine the toxic levels of the most active plant extracts using animal models.
1.9.3 Research questions
The study was guided by the following research questions
1. What medicinal plants are used to treat/manage HIV/AIDS immune compromised ailments?
2. What are the nutritive and medicinal values of the used plants?
3. What are the active chemical compounds responsible for bioactivity in the screened plant
extracts?
4. Are the plants raw materials safe for use as functional foods and pharmaceuticals?
13
2. Materials and methods
2.1 Description of study sites
This study was carried out in ‘Greater Mbarara’ which has been sub-divided into four districts of
Ibanda, Kiruhura, Isingiro and Mbarara. The districts are situated in western Uganda on
coordinates 00º 07´S 30 30E; 00
º 12´S, 31 00E; 00
º 50´S 30 50E and 00
º 36´S 30 36E
respectively (Fig. 1). The study areas are similar in climate, vegetation, population and economic
activities. They are in the same geographical range (U2), and have a mean annual rainfall
ranging from 1,100- 1,200 mm and temperatures ranging from 17ºC and 30
ºC.
The districts are predominantly occupied by Banyankore, Batagwenda, Bahima and other
immigrants; Bakiga, Baganda, Itesot, Banyarwanda, Congolese, and Bafumbira. Mbarara district,
the largest in the region, is bordered by Ibanda district in the north, Kiruhura district to the east,
Isingiro district to the south east, Ntungamo district to the south west, Sheema district to the west
and Buhweju district to the North West. Agriculture, which includes mixed farming, cultivation
of crops and grazing of cattle and goats, is the main economic activity in the districts and
accounts for more than 50% of the Gross domestic product. Crops grown include bananas, sweet
potatoes, Irish potatoes, maize, cassava and ground nuts. Health service provision is a key
priority of the districts. The districts have established health center facilities from health centre II
at Parish level, health center III at sub county level and Health Center IV at county level.
This study was done in western Uganda due to the highest burden of HIV/AIDS in the region
with a prevalence rate of 13.7% (UNGASS, 2010), and also due to a lot of biodiversity which is
beneficial to human health and has been used overtime by the local communities (Anoka et al.,
2008; Anywar et al., 2014; Asiimwe et al., 2013; Bunalema et al., 2014; Byarugaba et al., 2007;
Kakudidi et al., 2000; Kamatenesi-Mugisha and Oryem-Origa, 2007; Kamatenesi et al., 2008;
Katuura et al., 2007; Namukobe et al., 2011; Tabuti et al., 2003). Therefore, the rich biodiversity
forms a basis for the investigation on the biological active substances of natural medicines that
can pave way for the development of new alternatives and environment friendly medicines that
can be used in the management of HIV/AIDS opportunistic infections and other diseases, such
as, malaria, respiratory, cardiac and gastrointestinal ailments.
14
Figure 1. Location of study areas in western Uganda
15
2.2. Ethnobotanical field survey (Papers I, II, III)
An ethnobotanical study was conducted in four districts of western Uganda; Mbarara, Ibanda,
Kiruhura and Isingiro from December 2010 to May 2011. Key informants were selected
purposively (Ma Dolores, 2007; Martin, 1995) based on the skills, knowledge and practices in
medicinal plant usage. The key informants were renowned traditional medical practitioners such
as herbalists, traditional birth attendants and other local herbal medicine users like the elderly
women who were identified and chosen from the different villages with the assistance of local
administrators and community elders through visiting homesteads (Fig 2).
Fig 2. Methods used in field data collection
Subsequent interviewees were found with snow ball sampling (Salganik and Heckathorn, 2004).
In all the study areas, semi structured interviews with the aid of a questionnaire, focused group
discussions (FGDs) and field visits (Silverman, 2005) were used to document indigenous
knowledge on local names of plants used, parts and growth forms of the plants, conservation
status, mode of preparation and application of the herbal remedies. Interviews were conducted in
16
the local language of the area, Runyankore, except for a few cases where respondents were not
natives and translators were hired. Interviews were made with each traditional healer about the
knowledge and use of medicinal plant species used to treat human diseases in the study area,
with emphasis on opportunistic ailments associated with HIV/AIDS. The healers were
professional practitioners who medicate the local people by using crude extracts from plant
materials (leaves, bark, roots, seeds, flowers, stem and fruits). In order to comply with
internationally accepted ethical standards and ensure the protection of participants the proposal
for this study was approved by the Uganda National Council for Science and Technology. Before
conducting interviews, the aim of the study was explained clearly and informants were asked for
their consent. The purpose and benefits of the study were explained to the participants. The
questionnaire also included questions name of respondent, age, gender, religion, education, tribe
and occupation. The village, Parish, Sub-county and County where respondents came from were
also recorded.
The HIV/AIDS opportunistic conditions considered during the study were tuberculosis, herpes
zoster (shingles) and oral candidiasis. Other symptomatic but undefined conditions considered
were malaria, fungal infections of the skin, cough, diarrhoea, syphilis, hiccups, fever and
headache (CDC, 2013). Nutrition-related conditions such as anaemia, appetite and immunity
boosting were also considered. Plant specimens were identified in the field while those that could
not be identified were collected, pressed and deposited at Makerere University Herbarium for
identification. The specimens were identified by comparing with the already existing specimens
and using taxonomic literature such as (Katende et al., 1999; Maundu et al., 1999; Tallantire and
Lind., 1962). Scientific names of plant species were identified basing on the International Plant
Name Index (IPNI).
2.3 Selection of plants and extract preparation for bioactivity evaluation
One of the standard approaches for selecting plants for drug discovery is the use of ethnomedical
information about the traditional uses of the plants against various disease conditions (Fabricant
and Farnsworth, 2001). For this study, twenty plants that were frequently mentioned in all the
study areas were selected for bioactivity analysis. Secondly, there was little or no ethnobotanical
and ethnopharmacological information about the selected plants. One of the plants was selected
17
for curiosity purposes basing on its utilization by both humans and chicken believed to be a good
vegetable and changes the egg york to yellow. Lastly, some of the species from the same genera
as the selected plants elsewhere in the world have shown significant antioxidant and
antimicrobial activities.
Fresh plant materials (leaves and stem barks) were collected from peoples’ herbal medicine
gardens and from the wild in the various villages in Mbarara district. The plant materials were air
dried and ground into powder using a mortar and pestle, and finally stored in polythene bags
before analysis. The powders (100g each) were first soaked in ethanol for 48 hours, filtered and
dried using a rotary evaporator. The powders were also soaked in water and the extracts were
concentrated to dryness using an oven at 40ºC. The process was repeated for all the samples and
the extracts stored under room temperature for further analysis. Dried leaves and bark samples
(100g) were also subjected to steam distillation for isolation of essential oils using hexane which
was then evaporated using a rotary evaporator. The essential oils were transferred to clean vials
and stored in a freezer until analysis with Gas Chromatography – Mass Spectrometry.
2.4 Qualitative phytochemical analysis of the crude extracts
Phytochemicals are the core of phytomedicines; their therapeutic efficiency directly correlates
with the presence of various phytochemical compounds. In this study, phytochemical screening
was done to determine the groups of chemical compounds present in the water and ethanol plant
extracts. Chemical qualitative analysis for major active constituents such as alkaloids, tannins,
steroids, reducing sugars, coumarins, anthraquinones and carotenoids, was undertaken using
standard qualitative methods of analysis as described by (Harbone, 1993; Trease and Evans.,
2002).
2.4.1 The use of water and ethanol in phytochemistry
Since herbal medicine research aims at identifying bioactive phytocompounds in medicinal plant
extracts used by local people to treat diseases based on indigenous knowledge, the solvent
chosen must be the same as that used by local communities. Hence, water and ethanol are the
commonly used solvents. Water is a universal solvent used to extract plant products with
antimicrobial activity (Prashanti et al., 2011) and ethanol (at a concentration of 70-96%) is more
effective in isolating bioactive phytocompounds than water. Ethanol is used by local
18
communities in form of fermented porridge or local brew (made out of banana juice and
fermented sorghum – locally known as ‘tonto’ in Runyankore dialect) to extract and administer
the herbal remedies. Since this study aims at validating indigenous knowledge, water and ethanol
extracts were prepared for ethnopharmacological screening. Nearly all identified components
from plants active against microorganisms are aromatic and saturated organic compounds, and
are most often obtained through ethanol and water extraction (Lewis and Elvin-Lewis, 1995).
2.4.2 Chemical qualitative analysis of the aqueous and ethanol extracts
Identification of steroids / triterpenoids
The aqueous leaf extract (3 ml) was evaporated to dryness in a water bath; acetic anhydride (0.5
ml) and chloroform (0.5 ml) were added. Concentrated sulphuric acid (1 ml) was then added at
the bottom of the test tube containing the sample. The contact zone of the two liquids was
observed for a violet or brownish red ring formation.
Identification of basic alkaloids
The aqueous leaf extract (10 ml) was evaporated to dryness and hydrochloric acid (1.5 ml, 2%)
was added to the residue. The solution was then divided into three equal volumes in the test
tubes. Three drops of Mayer’s reagent was added to one test tube. Basic alkaloids were indicated
by a yellowish-white precipitate or coloration of the solution. The other test tube acted as a
control.
Identification of flavones aglycones
The aqueous leaf extract (3 ml) was evaporated to dryness in a water bath, the residue dissolved
in methanol (2 ml, 50%) and the solution heated on a water bath for 3 minutes. The solution was
then divided into equal volumes. A piece of magnesium ribbon (0.1g) and concentrated
hydrochloric acid (4 drops) were added to one test tube. Flavone aglycones were indicated by
change in colour of the sample to red or orange. The other acted as a control.
Identification of anthraquinones (Anthracenocides aglycones)
The aqueous leaf extract (3 ml) was transferred into a test tube and ammonia solution (1 ml,
25%) added. The solution was shaken thoroughly. Anthraquinones were identified by the
presence of red colour in the aqueous or ethanolic organic layer.
19
Identification of coumarins
The aqueous leaf extract (5 ml) was evaporated to dryness in a water bath. Distilled water (3 ml)
was added and the mixture heated on a water bath to boil and the mixture cooled under running
water. The solution (0.5 ml, 10%) was added. Both test tubes were observed under Ultra Violet
light and presence of coumarins was indicated by (blue or green) fluorescence in test tube
containing ammonia solution.
Identification of tannins
The ethanol leaf extract (1 ml) was diluted with distilled water (2 ml) and 3% solution of ferric
chloride (3 drops) was added. Tannins were identified by occurrence of a blackish- blue colour
for gallic tannins and green blackish for catechol tannins.
Identification of reducing compounds
The ethanol leaf extract (1ml) was diluted with distilled water (2 ml), and Fehling’s solutions 1
and 2 (1 ml) were added to the mixture. The solution was heated on a water bath. Reducing
compounds were identified by the presence of a brick red precipitate at the bottom of the test
tube.
Identification of flavonoids
The organic layer obtained after hydrolysis (5ml) was evaporated to dryness. The procedure was
then followed as earlier reported in identification of flavone aglycones in the water extracts as
indicated above.
Identification of sterol glycosides
The organic layer obtained after hydrolysis (5 ml) was evaporated to dryness. The procedure was
then followed as earlier reported in identification of sterols/ triterpenoids in the water extracts as
indicated above.
Identification of anthocyanins
Ammonia solution (3 ml, 10%) was added to acidic solution (3 ml) obtained after extraction of
the hydrolyzed solution drop wise. Change in colour of the solution from red to either violet or
greenish blue is due to presence of anthocyanins in the extracts.
20
2.5 Collection of headspace volatiles, Solid Phase Microextraction (SPME)
Solid phase microextraction (SPME) is a method used to collect headspace volatiles from plants
and insects (Borg-Karlson and Mozuraitis, 1996; Kännaste et al., 2008; Kännaste et al., 2013).
Fresh leaves (attached to the plant) and dry leaves were enclosed in bottles and the volatiles were
extracted by head space (HS) SPME using a Polydimethylsiloxae/divinylbenzene 65µm- fiber
(PDMS/DBV), equilibration time was 15-60 minutes. During collection of volatiles, leaves were
kept in a glass beaker that was sealed with aluminium foil (Fig 3). The extracted materials were
subsequently desorbed and analyzed in a gas chromatograph mass spectrometer (GC-MS). In
addition, the leaves were macerated in cold water for 1 hour and volatiles collected for 1 hr.
(A) (B)
Figure 3. SPME of volatiles: (A) fresh leaves;- (B) dry leaves
2.6 Isolation of essential oil
Fresh leaves, dry leaves and dry bark samples (100g) were each subjected to steam distillation
for 4-5 hours to isolate essential oils (Figure 4). The distillate was collected and extracted three
times with 100 ml of HPLC grade hexane (Carlo Erba Reagents, France). The hexane extracted
essential oil was dried using anhydrous magnesium sulphate (Alfa Aesar, United Kingdom),
filtered and the solvent was evaporated on rotary evaporator at 20º C under reduced pressure.
The essential oils were weighed and stored in tightly closed glass vials in a freezer at -20 ºC for
further analysis with GC-MS.
21
Figure 4. Steam distillation setup
2.7 Fractionation of essential oil using Medium pressure liquid chromatography
In order to separate compounds in the essential oil for antimicrobial activity testing, the oil of the
dry leaves of four plants (plants that showed significant antimicrobial activity) was subjected to
preparative medium pressure liquid chromatography (MPLC), equipment (Baeckstroem
SEPARO AB, Lidingö, Sweden) (fig 5). 20 g silica gel (60 Å, 40-63 µm, Carlo Erba Reagents,
France) was packed in 15mm internal diameter glass column fitted with pistons that could be
adjusted to the desired bed length of silica gel. An FMI lab pump, model QD (Fluid metering
Inc., Oyster bay, NY) was used to maintain constant flow and gradient of mobile phase. The
gradient elution was employed starting from 100% hexane till 20% ethyl acetate in hexane
running through 2.5%, 5% and 10% respectively. 100 ml of each binary solvent was used.
The eluent was collected in 10 ml fraction tubes that were analyzed by thin layer
chromatography (TLC) using pre-coated TLC plates (Silica gel 60 Å, 0.2 mm coating on
aluminum plates with F254). Based on TLC results, different fraction tubes were pooled together
giving rise to 5 fractions. The TLC plates were visualized using ultra violet light and vanillin
staining agent (3 g vanillin, 0.5 ml sulphuric acid in 100 ml absolute ethanol).
22
(A) (B)
(C)
Fig 5 (a) Baeckström SEPARO AB column (b) fractions eluted from column A (c) TLC plate for the
fractions shown in B
2.8 Chemical analyses of essential oils
Separation and identification of volatile compounds of essential oils and fractions were carried
out on gas chromatography – mass spectrometry (GCMS) using a Varian 3400 GC connected to
a Finnigan SSQ 7000 quadrupole mass spectrometer. The GC was equipped with a split/splitless
injector (splitless mode, 30 sec); the carrier gas was helium, with a constant pressure of 10 psi
and a flow rate of 1ml/min. The GC was equipped with a DB-WAX capillary column (30 m,
0.25 mm ID) and 25µm film thickness, J & W Agilent USA). The temperature program of the
GC oven was 40 ºC for 1 min, then increasing at a rate of 3 ºC /min till 235 ºC and maintained
for 9 min. The injector temperature was isothermally set at 230º C and the transfer line
connecting to the MS was isothermally set at 235 ºC. The MS ion source temperature was 150
ºC; mass spectra were obtained at 70 eV with a mass range of 30 - 400 m/z. Initial identification
of unknown compounds was made by comparing their mass spectra with NIST-08 (National
23
Institute of Standard and Technology, USA) MS library, Mass Finder 3, or comparing retention
index with published literature (Adams, 2007) or determined with reference to a homologous
series of normal alkanes. The final authentication was made by analyzing standard compounds
(purchased from Sigma-Aldrich) on GC-MS using same parameters that were used for essential
oil and fractions. One microliter aliquot containing 3µg/µl of essential oil or fraction was
injected into the GC injector for analysis.
2.9 Microorganisms and preparation of sample extracts used for antimicrobial assay
The antimicrobial activity of the plant extracts was tested against four standard human
pathogenic bacteria of the American Type Culture Collection (ATCC) viz, gram positive
Staphylococcus aureus (ATCC 25923), Streptococcus pneumoniae (ATCC 49619); gram
negative Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumoniae (ATCC 700603), and
two yeasts: Candida albicans (ATCC 10231) and Cryptococcus neoformans (ATCC 90112)
Ciprofloxacin (30 µg) and Nystatin (100 units) were used as positive standard controls for
bacteria and fungi, respectively. The organisms were obtained from MBN Clinical Laboratories,
Kampala, Uganda. The organisms were cultured on Mueller Hinton Agar for 24 hours at 37 ºC
and the fresh inoculums were used for the test against the leaf and bark extracts. Preliminary
screening was done on 20 selected plant species (Appendix C) and later bioassay guided
fractionation was done and four most active plants were selected for further screening;
Plectranthus amboinicus, Crassocephalum vitellinum, Erlangea tomentosa and Ipomea
hildebrandtii respectively (Appendix D).
2.10 Antimicrobial assay of the crude extracts (manuscript V)
The antimicrobial activity was measured by the agar well diffusion method (NCCLS, 2002;
Perez et al., 1990). Sterile nutrient agar was prepared and placed in labelled Petri dishes and
allowed to gel. The antifungal culture was prepared using the test organism on Sabourad
dextrose agar (SDA). Four wells were bored into the nutrient agar using a 7 mm sterile cork
borer and inoculums containing 105 CFU/ml of bacteria spread on the solid media with sterile
cotton swab moistened with bacterial suspension. Fifty microlitres (50 mg/ml) of essential oils
and fractions were dispensed in each well. The dried solvent extracts (0.2 g) were reconstituted
in 2 ml of normal saline and distilled water respectively, to make a stock solution with a
24
concentration of 100 mg/ml. Fifty microlitres (50 μl) of solvent extract of each plant was
dispensed into each well and allowed to diffuse for 30 minutes. Also 50 μl of normal saline was
placed in the wells separately as a control. Inoculation was done for 1 hour to ensure diffusion of
the antimicrobial agent into the medium. The extracts were screened in comparison with standard
Nystatin (100 units /disc) as a positive antifungal agent. Ciprofloxacin (30µg) was used as a
positive standard antibacterial agent while normal saline served as a negative control. Each test
was performed in duplicates and the inoculated petri dishes were incubated at 37 oC for 24 hours.
Antimicrobial activities were evaluated by measuring diameters of the inhibition zone of
microbial growth in millimeters (mm). Results are shown as mean diameters of minimum two
observations ± S.E.M.
2.10.1 Determination of Minimum Inhibitory Concentrations (MICs)
Minimum inhibitory concentration was carried out to determine the lowest concentrations of the
extract that will inhibit the visible growth of a microorganism under test after overnight
incubation. The estimate of minimum inhibitory concentration (MIC) was carried out by broth
dilution method. Half dilutions of plant extracts from 50 mg/ml to 1.56 mg/ml were used. Serial
dilution was carried out on the extracts using a 2-fold dilution factor, such that any dilution to the
right reduces the concentration by a factor of 2. The tubes were inoculated with microorganisms
suspension at a density of 105 CFU/ml. MIC values were taken as the lowest concentration of
extract inhibiting visible growth of test organisms after 24 hr of incubation at 37 ºC. The petri
dishes were incubated at 37 ºC for 24 hours.
2.10.2 Determination of Minimum Bactericidal (MBC)/ Fungicidal Concentrations (MFC)
The Minimum Bactericidal / Fungicidal Concentrations were carried out to determine the lowest
concentration of the extract required to kill a particular bacterium or fungus. For determination
of MBC/MFC, suspensions from the MIC studies were used. Using a standard loop of 6 mm,
samples from the MIC plates with visible growth were removed for subculture in Muller Hinton
Agar (MHA) and Sabaurouds dextrose agar. The isolated organism on the agar was incubated at
37 ºC for 18-24 hrs. After incubation, the plates were observed; the concentration that exhibited
no bacterial growth was considered as the MBC/MFC value (Williams and Wilkins, 2007).
25
2.10.3 Growth curve studies using Bioscreen method
The bacterial strains used in this experiment are: Staphylococcus aureus ATCC 25923 (+ve),
Streptococcus pneumonia, Pseudomonas aeroginosa (PA0-1) (-ve), Klebsiella pneumonia (-ve).
Klebsiella and P. aeroginosa were incubated overnight in 5% CO2 on Luria Agar Base, Miller
(DifcoTM) and they were suspended in Luria Broth (DifcoTM
). S. aureus was incubated
overnight in normal atmosphere on Tryptic Soy Broth (TSB), Y agar plates (1,5%
Bacteriological agar No 1 in Tryptic Soy Broth), (BactoTM
, Becton Dickinson, Sweden)
supplemented with 0.5% yeast extract (BBLTM
), and was suspended in (TSB) (BactoTM
)
supplemented with 0.5% yeast extract. S. pneumonia (T4) was incubated on Colombia base agar
(Acumedia, Baltimore, MD, USA) supplemented with 0.01% tryptophan (Merck, VWR
International, Sweden) and citrated horse blood (5%) in a 5% CO2 atmosphere, and it was
suspended in C+Y media.
Bacterial strains were streaked on their corresponding agar plates and incubated overnight in an
incubator with the required atmosphere. After 24 hours one loop full from each bacterial strain
was suspended in a tube with 10 ml of corresponding suspension media. The tubes were placed
in a water bath at 37 ºC until an optical density (OD) of 0.5, measured at 600nm was reached.
Using fresh tubes of media the cultures were diluted to an OD 600nm = 0.05 and 400 µl was
transferred to a Bioscreen C MBR multi-well plate (Oy Growth Curve AB Ltd). The growth
kinetics were monitored by changes in the OD 600nm at 37 ºC using a Bioscreen C (lab systems)
plate reader. When the OD 600nm reached approximately 0.3 this was considered time zero and 4
µl of the compounds of interest were added. Bacterial growth was monitored and measurements
were made every 20 min with 5 sec shake and 5sec rest cycle before every reading. The growth
was measured and recorded over a 12 hours period.
Viable counts:
The bacterial cultures were prepared as described before then at time point of interest 20 µl of
the bacterial culture was collected and added to 180 µl of 1xPBS so to achieve a ten-fold series
of seven concentrations. Using a multi-channel pipette, 10 µl of each of the seven concentrations
was plated onto a single agar plate. The plate was tilted at 45 degree angle whilst pipetting in
order to increase the surface area covered by each concentration. The agar plate was then
26
incubated over night at 37 ºC in the required atmosphere. Growth of bacteria was recorded in the
form of colony forming units (CFU).
3. Analysis of the plants for nutritive values (manuscript VI)
3.1 Evaluation of total antioxidant activity (TAC) by FRAP method
FRAP method depends on the reduction of ferric tripyridyltriazine complex to the ferrous
tripyridyltriazine by a reductant at low pH. The ferrous complex has an intensive blue color
which can be monitored at 593 nm (Benzie and Strain, 1996).
Reagents: acetate buffer, 300mM/L, pH 3.6 (3.1g sodium acetate 3H2O and 16 mL conc.;
Acetic acid per 1L of buffer solution); 10 mM/L TPTZ (2, 4, 6-tripyridyl-s-triazine) in 40 mM/L
HCl; 20 mM/L FeCl36H2O in distilled water. FRAP working solution: 25 mL acetate buffer, 2.5
mL TPTZ solution and 2.5 mL FeCl3 solution. Aqueous solution of known Fe (II) concentration
was used for calibration, in a range of 0.1- 0.8 mM/L. For the preparation of calibration curve
0.1, 0.2, 0.4, 0.6, 0.8 μM/mL aqueous Fe(II) as Mohr salts solution (1mM) were mixed with 2.5
mL FRAP working solution; FRAP reagent was used as blank. A concentration of 50 mg/ml of
extract was used. The absorption was read after 10 minutes at 25 °C and 593 nm. All
determinations were repeated three times. Total antioxidant capacity in Fe (II) equivalents was
calculated. Correlation coefficient (r2) for calibration curve was 0.9994.
3.2 Evaluation of total antioxidant activity (TAC) by DPPH method
The total antioxidant capacities (TAC) or the amount of specific antioxidants in different plant
extracts was measured using the DPPH (2, 2 – Diphenyl 1-1 picryl hydrazyl) assay which is
associated with electron and radical scavenging activity. The assay makes use of a stable free
radical, (1, 1 – diphenyl -2- picryl hydrazyl). DPPH shows a strong absorption maximum at
517nm (purple). The reaction involves colour change from purple to yellow followed by the
formation of DPPH upon absorption of hydrogen from an antioxidant. The degree of colour
change will be correlated with the samples’ antioxidant concentrations. Total Antioxidant
activity (TAC) was determined using Ultra Violet spectrophotometric method (Habila et al.,
2010; Nickavar et al., 2006).
Hydrogen atom – or electron-donation ability of the corresponding medicinal herbs was
measured from the bleaching of the purple-colored ethanol solution of DPPH. 0.5 mL of various
27
ethanol extracts diluted 1/10 were added to 2.5 mL of a 1 mM ethanol solution of DPPH. After
40 minute incubation at room temperature the absorbance was read against a blank at 517 nm.
Antioxidant capacity expressed as percentage inhibition of the DPPH free radical was calculated
in the following way (Burits and Bucar, 2000; Cuendet et al., 1997).
TAC DPPH (%) = (Ablank – Asample) /Ablank x 100 where A is absorbance.
3.3 Mineral nutrient analysis using atomic absorption spectrophotometry
Minerals are important for maintaining good health. Ten plants, selected on the basis of use in
traditional medicine for improving nutritional status, were analyzed for the presence of mineral
elements like zinc, iron and selenium, which are particularly important for HIV infected people
for regeneration of CD4 T cells and maintaining the sero status of patients (Mehta and Fawzi,
2010). About 1-2 grams of plant leaves was weighed into a clean dry silica dish/ crucible. Then
1.5ml of 15% magnesium acetate was added and the mixture heated on a hot plate to completely
char the sample. The crucible was placed in a muffle furnace maintained at 450 oC and allowed
to ash for 4 -6 hrs. The crucible was removed from muffle and placed in a desiccator and allowed
to cool at room temperature. The sample was diluted to get the mineral into the linear
concentration range of instrument using deionized water. The concentrations of iron, zinc and
selenium were determined by using atomic absorption spectrophotometer (Schimadzu – AA-
6300) according to AOAC official methods (AOAC, 2000). The reference standards were
prepared by suitable dilutions of the stock standard solutions of each micronutrient standard
solution.
3.4 Safety evaluation of selected plants (Paper IV)
3.4.1. Acute toxicity studies
Toxicity tests show how potent a chemical or substance is by measuring the concentration that
will affect the test organism. The aim of acute toxicity test is to establish a starting dose in
humans and the therapeutic index, i.e the ratio between therapeutically effective dose and the
lethal dose (LD50/ED50) (Gosh, 1984). The higher the index the safer the compound. LD50 test is
used to evaluate toxicity by determining the dose required to kill 50% of the test organisms. The
acute oral toxicity tests on plant extracts were carried out using female and male albino mice
28
(Lorke, 1983) and were conducted according to the Organization for Economic Cooperation
Development (OECD, 2002) guidelines 423 where the limit test dose of 5000 mg/kg was used.
Dose is the amount of test substance administered at one time, and is expressed as weight of test
substance per unit weight of test animal (mg/kg). Healthy young animals were acclimatized to
the laboratory conditions for at least 10 days prior to the test before being assigned to the
treatment groups. This was to ensure that the micro-environment of the laboratory was not a
factor to affect the tests. The animals were fasted overnight ( 18 hours) and divided into one
control group and three treated groups, each group consisting of two animals (male and female)
labeled I – IV. The different groups of mice were treated with different doses of the extracts
using the up and down procedure (OECD, 2008a). The mice were carefully observed for any
behavioral changes and toxicity manifestations at different time intervals. Control animals
received 1ml of distilled water through the same oral route using intragastric syringe. Extracts
that did not kill any mice were considered non-toxic. The volume of extract administered to the
mice was calculated as follows (Gosh, 1984): Volume of extract (ml) = weight of mice (kg) x
dose rate (mg/kg) / Stock solution (mg/ml)
3.4.2. Sub acute toxicity studies (Paper IV)
The sub acute toxicity test was performed following the Repeated-dose oral toxicity protocol
described by the OECD guideline 407 for testing of chemicals (OECD, 2008b). Twenty four
adult albino wistar rats were divided into four groups of 6 rats per group. Having determined that
the LD50 was above the limit test dose of 5000 mg/kg, the extract doses for sub acute test were
determined from ½, ¼ and 1/8 of the limit test dose of 5000 mg/kg. Groups I, II and III received
625, 1250 and 2500 mg/ kg body weight respectively, of the aqueous extract once daily for 28
days. Group IV (control) received 1 ml of distilled water. The body weights of individual
animals were evaluated weekly and recorded. The body weight changes were monitored
throughout the experimental period on weekly basis. Similarly, the animals were observed for
any manifestation of toxicity and mortality. At the end of 28 days, the rats were sacrificed by
choloform anesthesia. The liver, kidney, intestines and lungs were removed and preserved in
10% formalin. Tissue organs were sectioned in paraffin wax and stained with hematoxylin and
eosin for assessment of tissue morphology. Histopathological, hematological and biochemical
29
studies were performed to determine if there was any deleterious effect of the plant extracts on
the tissues and organs after extract administration for 28 days period of observation.
3.5 Statistical analysis of data
3.5.1 Quantitative analysis of ethnobotanical data (Papers I, II, III)
The therapeutic indications cited during ethnobotanical field interviews were grouped by body
systems according to the disease categories proposed by the International Classification of
Diseases (CDC, 2013; WHO, 2006). By means of the consensus of the informants, the
importance of a species for a determined purpose and the categories that present greater
importance to the people was obtained by the calculation of the Fidelity level (FL) and from the
Informant Consensus Factor (FIC) indices respectively (Friedman et al., 1986; Heinrich et al.,
1998). To test the consistency or homogeneity of informant’s knowledge in treating a particular
illness category, calculation of consensus factor (Fic) was followed (Heinrich et al., 1998; Trotter
and Logan, 1986). This factor is given as: Fic = (Nur – Nt) / (Nur – 1); Where Nur is the
number of use reports of informants for particular ailment category, Nt is the number of species
used for a particular ailment category by all informants. This factor ranges from zero to one,
where increasing values indicate high rate of informant consensus. To assess the importance of
individual species in each group, fidelity level was calculated (Friedman et al., 1986). This
shows the percentage of informants claiming the use of a plant species for the same major
purpose. Fidelity level index, FL = Ip/N x100, where Ip is number of informants who indicate use
of a species for the same major ailment, N is the total number of informants who mentioned the
plant for any other use (Friedman et al., 1986). The calculation of FL was limited to plants with
at least more than 5 citations for a particular illness category. Increasing values of FL for a
species indicate its uniqueness to treat a particular illness (Pandikumar et al., 2011).
3.5.2 Analysis of toxicity results
The data for toxicity tests was subjected to one-way analysis of variance (ANOVA) test and
differences between the control group and extract treated groups were determined by post hoc
Dunnett’s multiple comparison tests, using Graph Pad Prism (Version 5) software to obtain the
toxicity levels of the different plant extracts. Results were presented as mean ± SEM and
considered to be statistically significant at p ≤ 0.05 with 95% confidence interval.
30
4. Results and discussion
4.1 Ethnobotanical studies - documentation of nutri-medicinal plants used in the
management of HIV/AIDS opportunistic ailments in western Uganda (Papers I, II, III)
The purpose of this study was to document traditional botanical knowledge of 4 study areas and
to assess the quantitative comparison of the medicinal plant knowledge of the local communities
using informant consensus factor (ICF), Fidelity Level (FL) and Use values (Heinrich et al.,
1998; Trotter and Logan, 1986). The results presented in this chapter are the outcome of a series
of interviews conducted between December 2010 and May 2011 with local communities of
Mbarara, Ibanda, Isingiro and Kiruhura districts in western Uganda. This chapter attempts to
provide answers to the following research questions; what medicinal plants are used to
treat/manage HIV/AIDS opportunistic infections? What are the most important plant use
categories? What are the most useful species per use category? Which plant families contain the
highest number of useful species in each use category? In order to evaluate the usefulness of
different plant species to the local communities, different plant species and their uses are
discussed in two broad categories; medicine and food (Papers I, II, III). In the context of this
study, a response or citation is defined as an answer from a participant with regard to the use of
particular plant species. A plant use is a well defined use of a particular plant species for one
particular goal by one or more participants. Apparently plant use and ethnobotanical
relationships continue to increase with increase in biodiversity inventories and associated
attributes to particular taxa up to species level.
After getting prior informed consent, interviews were conducted in order to make informants
mention the medicinal plants that they use in the management of opportunistic infections of
HIV/AIDS. A total of 143 informants, including 114 females and 29 males aged between 23 and
85 years participated in the study. Forty nine (34.3%) traditional medical practitioners
(traditional healers and traditional birth attendants) were interviewed. The selected respondents
especially traditional healers were well known in the community due to their long practice in
service provision related to traditional health care. This study recorded 324 nutri-medicinal
plants used in the management of opportunistic ailments of HIV/AIDS and other diseases, 51 of
which were not properly identified. The plants are distributed in 75 Families. Based on the
31
information obtained, the reported HIV related diseases were grouped into 5 major categories
(Paper I, II). The categories with high number of plants were respiratory infections (cough,
tuberculosis), appetite and immunity boosting as well as bacterial and fungal infections (candida,
syphilis, UTIs etc). Therefore, the results of the ICF point to these medicinal categories as the
ones that informants are most confident about. High ICF values are obtained when only one or
few plant species are reported to be used by a high proportion of informants to treat a particular
ailment. Hence, high ICF values are used to select interesting species for the search of bioactive
compounds (Canales et al., 2005; Heinrich et al., 1998). Research shows that plant species with
higher frequencies of use among cultural groups contain higher levels of secondary alkaloids
than those with lower frequency of use (Quinlan et al., 2002). The ICF results could be useful in
prioritizing medicinal plants for scientific validation of plants and plant products as
pharmacologically effective remedies are expected from plants with higher ICF values.
However, the study also recorded plants used for other disease conditions other than HIV/AIDS
opportunistic infections. The ailments were grouped into 14 categories, with the highest number
of plants being used for gastrointestinal and reproductive health disorders (Paper III). Most of the
plants used had more than a single therapeutic use, for instance Albizia coriaria was used for
herpes zoster, cough, skin infections, tuberculosis, syphilis, headache, diarrhoea and uterine
pains. On the other hand, one ailment would be treated using many plants, for instance, cough
was treated using Warburgia ugandensis, Albizia coriaria, Basella alba, Cajanus cajan,
Tetradenia riparia, Abutilon guineense and Acacia hockii.
The healing potential of the most frequently reported plants was determined using fidelity level
(FL) (Friedman et al., 1986) (Papers I, II, III). An increasing value of fidelity level for a species
indicates its uniqueness to treat a particular illness. Families Asteraceae, Fabaceae,
Euphorbiaceae and Lamiaceae were the most dominant throughout the study areas (Fig 6).
Family Asteraceae had the highest number of plants used (44), followed by Fabaceae (20),
Lamiaceae (19) and Euphorbiaceae (18) respectively.
32
Fig 6. The most commonly utilized plant families
Majority of herbal remedies were prepared by boiling, grinding /pounding, soaking in hot or cold
water of fresh or dry plant materials (leaves, roots, seeds, fruits and flowers). Some herbal
preparations were taken by mixing with food, honey, milk or tea, especially for oral
administration. Other drugs were applied eternally for conditions like fungal infections of the
skin. It was reported that dosages were given by estimation depending on age and type of
sickness. About 61% of herbs are used for remedy preparation, followed by trees and shrubs.
Throughout the study areas, more than half of nutri-medicinal plants grow in natural
environments including forested areas, fallow and farm lands. This puts the key plant species at a
risk since they are threatened by human activities. Hence, there is a need for in-situ conservation
and domestication of key plant species. The only constraint remains with plant species that
require a unique micro-climate if they are shade or non-shade plant species.
This study follows a quantitative approach because it quantifies useful plant species and is based
on participant consensus. Quantitative Ethnobotany has been defined as the application of
quantitative techniques to the direct analysis of plant use data (Phillips and Gentry, 1993a).
Ethnobotanical research is currently experiencing a revival as evidenced by numerous recent
publications (Abbasi et al., 2013; Anywar et al., 2014; Asiimwe et al., 2013; Bunalema et al.,
2014; Byarugaba et al., 2007; Claudio et al., 2013; Guimbo et al., 2011; Gurib-Fakim, 2006;
Hardon et al., 2008; Hernández et al., 2003; Kakudidi et al., 2000; Kamatenesi, 2010; Kazhila
and Marius, 2010; Padhi and Panda, 2013; Senthilkumar et al., 2013; Traore et al., 2013; Wu et
33
al., 2001; Xi-long et al., 2013). Plants used in traditional medicine therefore have an important
role to play in the maintenance of health in all parts of the world and in the introduction of new
treatments. This study revealed a rich diversity of nutri-medicinal plants used to treat various
disease conditions.
4.2 Phytochemistry, antimicrobial and antioxidant activities of nutri-medicinal plants
(Manuscripts V & VI)
4.2.1 Chemical analyses of essential oils
The importance of ethnomedicinal plants lies not only in their chemotherapeutic value in
traditional health care but also in their potential as sources of biologically active entities.
Phytochemistry is the core of phytomedicines because therapeutic efficiency correlates directly
with the presence of various phytochemicals. The aim of this study was to evaluate the
phytochemical profiles of selected nutri-medicinal plants, and their biological (antibacterial,
antifungal and antioxidant) activities. Ten plants were selected for bioactivity testing based on
ethnomedical information provided by local communities and traditional medical practitioners,
and how frequently they are used to treat opportunistic infections of HIV/AIDS (Paper I and II).
Qualitative chemical analysis revealed the presence of secondary metabolites: tannins, saponins,
alkaloids, flavonoids, terpenes and terpenoids, coumarins, reducing sugars and steroids. The
pharmacological properties of these groups of chemical compounds are reported in literature
(Ango et al., 2012; Cowan, 1999; Teke et al., 2013). For instance, tannins and flavonoids have
antioxidant, anticancer, antifungal, antiviral, antiallergic and detoxification activities (Adedapo,
2013). Their presence in the leaves of the studied plants justifies their use in traditional medicine
to treat candida, syphilis, boost immunity and treat many other infectious diseases. The water
extracts were rich in phytochemical compounds especially tannins, saponins and steroid
glycosides. This justifies the use of water as the main media for herbal remedy preparation.
Some of the main compounds identified by GC-MS include linalool, linalyl acetate, β-cubebene,
carvacrol, α- terpineol and γ-terpinene. The biological and medicinal properties of the same
compounds have also been reported by other researchers (Paduch et al., 2007; Skočibušić and
Bezić, 2004; Thanighaiarassu and Sivamani, 2013).
34
It is well known that herbal medicinal products usually contain more than one plant or active
constituents and their therapeutic efficacy is not provided by a single compound. Some of these
compounds act synergistically to modify the bioavailability and efficacy of the active
constituent. For instance, tannins have been considered in traditional medicine to treat various
diseases, and their synergistic effects with various antibiotics, such as carbenicillin and
tetracycline, have been proved beneficial against antibiotic resistance bacteria (Dusane et al.,
2015; Okuda and Ito, 2011).
4.2.2 Major groups of volatile compounds identified in the essential oils
Medicinal plants have been a source of a wide variety of biologically active compounds for
centuries and used extensively as crude material or as pure compounds for treating various
disease conditions. There is ongoing research to explore the diverse sources that may provide
more effective and less toxic antimicrobial compounds (Saleem et al., 2010). Figures 7- 10 show
the major compounds present in the essential oils of the studied plants.
Fig 7. Major compounds in the essential oil of Plectranthus amboinicus
35
Fig 8. Major compounds in the essential oil of Crassocephalum vitellinum
Fig 9. Major compounds in the essential oil of Erlangea tomentosa
Fig 10. Major compounds in the essential oil of Ipomea hildebrandti
36
4.2.3 Antibacterial and antifungal testing of plant extracts (Manuscript V)
Microbial infections are the most frequent opportunistic diseases occurring during HIV/AIDS
which affect many people in Africa. The spread of drug resistant pathogens is one of the most
serious threats to successful treatment of microbial diseases. In response to this, there is an
increasing interest in the use of essential oils and plant extracts as sources of natural products
which have been screened for potential uses as alternative remedies for the treatment of many
infectious diseases (Tepe et al., 2004). Studies have shown that essential oils possess
antibacterial, antifungal and antioxidant properties (Burt, 2004). The number of higher plant
species on this planet is estimated to be between 250,000 – 500,000 of which only 6% have been
screened for biological activity and a reported 15% have been evaluated phytochemically
(Verpoorte, 2000). Plant extracts have been of great interest due to their potential uses as
alternative remedies for the treatment of many infectious diseases particularly as antibiotics.
Plant-based anti-microbials represent a vast untapped source of medicines with therapeutic
potential (Cowan, 1999). In the present study, four plants that are traditionally used to treat
various ailments including HIV/AIDS – related infections were selected for antimicrobial testing
(Table 1).
Table 1. Traditional uses of the plants tested for antimicrobial activity
No. Scientific name Local name
(Runyankore)
Ailment (s) treated Parts
used
Family name
1 Plectranthus
amboinicus (Lour)
Spreng.
Akacuncu
akakye
Cough, immunity boosting,
colic and stomach infections,
skin infections
Leaves Lamiaceae
2 Crassocephalum
vitellinum S. Moore
Esunuunu Herpes zoster, fever, diarrhoea,
oral candidiasis, syphilis,
energy boosting, tumors,
uterine pains, headache
Leaves Asteraceae
3 Erlangea tomentosa
S. Moore
Ekyoganyanja Skin infections, diarrhoea,
syphilis, appetite boosting,
anaemia,
Leaves Asteraceae
4 Ipomea hildebrandti
Vatke
Bingirebita Tinea capitis and corporis and
other fungal infections of the
skin, tumors, herpes zoster and
syphilis,
Leaves Convolvulaceae
37
Two tests were used to evaluate the antimicrobial activities of essential oils. In the first test, the
agar well diffusion method was used to evaluate the antimicrobial activity by measuring the
inhibition zone against the test microorganisms. The activity of essential oils and fractions,
water and ethanol extracts of the four plants were tested against 2 gram negative, and 2 gram
positive opportunistic human bacteria, and 2 fungal pathogens using agar well diffusion method.
All the tested extracts were active against one or more of the organisms tested (Appendix C).
The findings of antimicrobial screening showed that some of the essential oil fractions were
more active than the pure oils. This is because fractionation of the crude extracts allows the
distribution of these metabolites in the fractions according to polarity (Houghton and Raman,
1998). Hence, the phytocompounds can easily be separated and the associated activity might be
due to the presence of different compounds in the individual fraction.
The essential oil from dry and fresh leaves from Plectranthus amboinicus was screened to
compare the activities of compounds from both forms of leaves. The essential oil from the fresh
and dry leaves of Plectranthus amboinicus was found to have strong antifungal activity against
Candida albicans (MIC= 1.56 mg/ml) and Cryptococcus neoformans (Manuscript V). Previous
studies on the methanolic extract of P. amboinicus have indicated that the plant has antifungal
activities against Candida albicans and Cryptococcus neoformans (Nagalakshmi et al., 2012).
The essential oils and fractions, water and ethanol extracts of other plants were also tested
against the same organisms (Appendix C & D). The activity of the water extracts was very poor.
This is also reported by other researchers that organic solvents and essential oils give more
consistent biological activity compared to the water extracts (Prashanti et al., 2011). In this
study, only the bark aquoeus extracts of Albizia coriaria and Maytenus senegalensis showed
mild activity against Streptococcus pneumoniae and Staphylococcus aureus. The essential oil
and fractions of Crassocephalum vitellinum also showed very strong activity against
Cryptococcus neoformans and Candida albicans. The fractions were more active on the yeasts
than the pure oil. The study showed that on further fractionation of the essential oils of
Crassocephalum vitellinum the hexane soluble fractions demonstrated the highest sensitivity
towards Cryptococcus neoformans. However, the water and ethanol extracts showed weak
activity against the tested bacterial strains. Similar findings on C. vitellinum showed that the
38
ethanolic extract exhibited weak antibacterial activity (Moshi et al., 2014). Hence, the plant has
displayed good antifungal activity. Similar studies on other species, such as, Crassocephalum
bauchiense showed that the ethyl acetate extract was active on Candida albicans and
Cryptococcus neoformans than the fractions (Mouokeu et al., 2014). The antifungal properties of
C. vitellinum can be attributed to the presence of tannins, terpenes, flavonoids, phenols and
sterols and which have been reported in literature (Cowan, 1999; Paduch et al., 2007). These
results support its traditional use in treatment of oral Candidiasis (Table 1).
The pathogen Cryptococcus neoformans is an environmental fungal pathogen afflicting
immunocompromised patients as well as immune competent individuals. Infection occurs in the
upper respiratory tract where pathogenesis presents as cryptococcal meningitis, causing life
threatening serious opportunistic infection meningoencephalitis (Samie et al., 2010; Tripathi et
al., 2012). Research shows that C. neoformans is a major threat to AIDS patients in Sub Saharan
Africa, causing illness to about 1 million people annually, despite major developments in HIV
treatment (Park, 2009; Warkentien and Crum-Cianflone, 2010). About 5-8% of HIV positive
patients experience the disease during the course of AIDS. Cryptococcosis is observed in
advanced stage when HIV patients have CD4+ T cell count of < 50 cells/ µl of blood (Sandhu
and Samra, 2013). Research also indicates that C. albicans and C. neoformans are some of the
causes of diarhoea in HIV positive children (Ukaropol, 2003). Candida albicans is a yeast
infection, which in immunocompromised conditions may affect the skin, oral mucosa or genital
areas.
The results obtained from this study support the traditional uses of the plants for treating
opportunistic infections in HIV/AIDS patients. These results are relevant since C. albicans and
C. neoformans are the leading causes of fetal fungal infections in immunocompromised patients.
The essential oil from the fresh leaves of P. amboinicus was also active against Klebsiella
pneumoniae compared to the standard ciprofloxacin (Appendix D). Figure 11 shows the activity
of the essential oil extracts of the plants against different organisms tested. P. amboinicus was
active against all the test organisms, while Ipomea hildebrandti was only active against Candida
albicans and Cryptococcus neoformans. This confirms its use in traditional medicine to treat
39
fungal infections especially tinea infections (Table 1). Results also indicate that all the essential
oils were active against C. albicans and C. neoformans (Fig 11).
Fig 11. Activity of the essential oils against the tested organisms
4.2.4 Growth curve studies using Bioscreen method
In the second test, the Bioscreen method was used to measure microbial growth (Johnston, 1998;
Wu et al., 2000). Growth pattern of the test organisms was studied on the basis of optical density
(OD) and log10 cfu/ml. From the study results, only the crude essential oil of Plectranthus
amboinicus showed activity against Klebsiella pneumoniae (Fig 12) and Pseudomonas
aeruginosa (Fig 13). With an increase in the antibiotic resistance over the past decade there has
been a renewed interest in alternatives to antibiotic therapy. For instance, Pseudomonas
aeruginosa, an opportunistic human pathogen that causes cardiac and respiratory infections in
cystis fibrosis patients and in patients with compromised defense mechanisms, is resistant to
various antibiotics. This has led to the search for new therapeutic strategies (Cooper et al., 2011).
In this study, P. amboinicus was found to be active against P. aeruginosa. This could be due to
the high amount of carvacrol present in the essential oil. This may be supported by research
which shows that carvacrol inhibits the growth of several bacterial strains including P.
aeruginosa by causing damage to the cell membrane of the bacteria and inhibiting their
proliferation (Di Pasqua et al., 2007). The studied plants showed promising results as
antibacterial and antifungal potential drugs.
40
Fig 12. Activity of essential oil of P. amboinicus against Klebsiella pneumoniae
Fig 13. Activity of essential oil of P. amboinicus against Pseudomonas aeruginosa
Many plant-derived compounds such as terpenes have been found to be active against a variety
of microorganisms, including gram positive and gram negative bacteria (Paduch et al., 2007).
Linalool, one of the major components in this study has been found to have antimicrobial activity
against various microbes and inhibits spore germination and fungal growth (Josphat et al., 2007).
A mixture of α-terpineol, 1, 8-cineole and linalool has been shown to possess antibacterial
activity against gram negative and gram positive bacteria of the oral cavity, skin and respiratory
tract (Paduch et al., 2007). The presence of similar compounds in this study may support the use
of the plants under study to treat candidiasis and other fungal infections of the skin. Structures of
some terpene compounds with antibacterial and antifungal activity are listed in Appendix B.
41
4.2.5 Antioxidant capacity and mineral nutrient composition (Manuscript VI)
Antioxidants are vital substances which possess the ability to protect the body from damages
caused by free radical-induced oxidative stress, whereas the micro- and macro minerals are the
key components for overall body growth and development. Natural antioxidants present in plants
are responsible for inhibiting or preventing deleterious consequences of oxidative stress, which
play a role in the pathogenesis of opportunistic infections of HIV/AIDS (Otang et al., 2012). For
instance, terpenoids have been found to have immunomodulatory properties (Theis and Lerdau,
2003).
Ten plants were screened for antioxidant activities and mineral nutrient composition. The
antioxidant activity was determined using DPPH and FRAP assay tests. Reducing power assay is
used to evaluate the ability of natural antioxidant to donate an electron and DPPH assay is a
measure of the presence in the medicinal plants of the compounds with radical scavenging
capacity. The extracts expressed strong antioxidant power as measured by DPPH (free radical
scavenging) compared to FRAP (reducing power). According to their antioxidant capacity, the
10 medicinal plant extracts were divided into 3 groups; a) very low FRAP (< 1 mML), n= 3; low
FRAP (1-5 mML), n= 5; good FRAP (>5 mML), n=2. Symphytum officinale and Erlangea
tomentosa showed promising antioxidant capacity as reducing power. The highest TACDPPH
values were obtained for Pseudarthria hookeri, Plunchea ovalis and Ipomea hildebrandti. The
antioxidant activity of other Plunchea species has been reported (Hidayat et al., 2013). A survey
on the genus Plunchea revealed the presence of eudesmane – type of sesquiterpenoids,
monoterpenes and flavonoids (Goyal and Aggarwal, 2013; Hidayat et al., 2013). Similary,
several compounds belonging to the groups of monoterpenes and flavonoids have shown
anticancer activity, which is due to the antioxidant activity. This study showed that P. ovalis had
38% scavenging activity. This could be attributed to the presence of high amounts of flavonoids
and tannins in the plant extracts.
Plant antioxidants are composed of a number of different substances like ascorbic acid,
terpenoids and polyphenolic compounds such as flavonoids. Therefore, the presence of these
phytochemicals could support the herbal medicine uses of the plants as antioxidants whose
antioxidant activity is well known (Gonzalez-Burgos and Gomez-Serranillos, 2012; Graßmann,
42
2005; Ruberto et al., 2002). Since reactive oxygen species are thought to be associated with the
pathogenesis of AIDS, and HIV-infected individuals often have impaired antioxidant defenses,
the inhibitory effect of the extracts on free radicals may partially justify the traditional use of
these plants in the management of opportunistic infections in HIV/AIDS patients. Many plants
produce a variety of compounds including terpenes and their derivatives which have been found
to be useful in the prevention and therapy of several diseases including cancer and to have
antioxidant and immunomodulatory properties (Gonzalez-Burgos and Gomez-Serranillos, 2012;
Ruberto and Baratta, 2000; Ştef et al., 2009). Such compounds protect body cells against
damaging effects of reactive oxygen species by scavenging the free radicals in the body.
The plants were analyzed for the presence of zinc, iron and selenium, which are particularly
important for HIV infected people, mainly for regeneration of CD4 T cells, reducing diarrhoeal
morbidity and maintaining the sero status of patients. The results for mineral nutrient analysis of
the plants are given in manuscript VI. The plants were selected on the basis of use in traditional
medicine for improving nutritional status in terms of immunity and energy boosting. The mineral
contents in the plant samples were found at different levels, ranging from 0.03 – 6.9 mg/kg dry
weight. According to research, the amounts of minerals present in the studied plant species meet
the average dietary intake level that is sufficient to meet the nutrient requirements needed for
daily maintenance of the immune system (Manuscript VI).
4.3 Safety evaluation of nutri-medicinal plants ( Paper IV)
4.3.1 Acute toxicity study
Toxicity is the ability of a chemical to damage an organ system, such as liver or kidneys; to
disrupt a biochemical process such as the blood-forming mechanism, or disturb an enzyme
system at some site in the body. Toxicity studies help to assess the potential health risks caused
by intrinsic adverse effects of chemical compounds present in plant extracts. However, all
substances have some level of toxicity above the allowable limit which is dosage. There are two
types of toxicity, acute and chronic. Acute toxicity is the adverse effects occurring within a short
time of oral administration of a single dose of a substance or multiple doses given within 24 hrs
(OECD, 2002). A chemical is considered to be extremely toxic if it has LD50 of 1mg/kg and
practically non toxic if it has an LD50 of 1500 mg/ kg and above (Gosh, 1984).
43
Toxicity tests were done in mice and rats to determine the safety of the plant extracts. The oral
route of administration of the extracts to the test animals was used, which is in line with the route
of administration of medicines by humans. The study was conducted to determine whether crude
plant extracts of selected nutri-medicinal plants are safe for human consumption, since most of
the remedies are administered orally. Toxicological assessment of the water, ethanol and
essential oil extracts of Plectranthus amboinicus, Pseudarthria hookeri, Tarenna pavettoides,
Symphytum officinale and Plunchea ovalis was carried out in albino mice and rats. Acute toxicity
was tested in mice starting with doses of 2000 mg/kg up to a high concentration of 10,000 mg/kg
body weight, when no toxicity signs or death occurred and dose was increased sequentially. As
the dose increased, signs of acute toxicity became more obvious especially with essential oils
where several deaths were recorded (Appendix E). The water and ethanol extracts did not show
any adverse signs of toxicity or cause any mortality, but there was increased urination,
defecation, drowsiness, twitching of gut muscles and hypoactivity. The lethal dose at 50 %
(LD50) for some of the essential oils (Table 2 and Fig 14), water and ethanol extracts was greater
than 5000 mg/kg for a single extract. Probit analysis was used to estimate LD50 value (Finney,
1952). The extracts could therefore be classified as being safe following the OECD guidelines
for acute oral toxicity (OECD, 2002).
Table 2. Results of lethal doses of essential oil of Pseudarthria hookeri for determination of LD50 after
oral administration in mice (n=6)
Group Dose (mg/kg) Log dose No: dead % Dead Probits
1 3,500 3.54 0/6 0 3.04
2 4,500 3.65 1/6 16.7 4.18
3 5,500 3.74 2/6 33.3 4.75
4 6,500 3.81 3/6 50.0 5.15
5 7,500 3.87 6/6 100.0 6.96
Log LD50 = 3.74, antilog of 3.74= 5506 (Fig 14). Hence, LD50 is 5506 mg/kg. According to
OECD guidelines and Gosh, (1984) classification of toxic doses, whereby LD50 above
5000mg/kg is considered to be experimentally safe.
44
Fig 14. Plot of log doses versus probits from table 1 –for calculation of LD50
4.3.2 Sub acute toxicity studies (Paper IV)
Sub- acute toxicity test was done in rats using standard procedures (Gosh, 1984; Lorke, 1983;
OECD, 2002). The purpose of this test was to determine the maximum tolerated dose and nature
of toxic reactions. In sub acute toxicity tests, the adverse effects may manifest in form of
alterations in levels of biomolecules such as enzymes and metabolic products, normal
functioning and histomorphology of the organs (Ashafa et al., 2009). Results of such tests in
animals can then be extrapolated onto human beings. However, man is generally six times as
sensitive as the dog, and ten times as sensitive as the rat to the toxic effects of drugs (Gosh,
1984). However, metabolic variations have to be put into account. Special tests may be done to
determine teratogenic effects of the drug and also whether the chemical is carcinogenic or not
(Gosh, 1984).
The sub acute toxicity test was evaluated through biochemical, hematological and
histopathological parameters. Daily doses of 2500 mg/kg, 1250 mg/kg and 625 mg/kg of the
aqueous extract were administered for 28 days and signs of toxicity were recorded.
Histopathological examination was done on the liver, kidneys, lungs and intestines. Body weight
changes were measured weekly for 28 days of daily single dose of extract administration (Paper
IV). The results of sub acute test showed that Plectranthus amboinicus is not toxic, LD50 >
10,000mg/kg, which is above the limit test dose of 5000 mg/kg. There was a significant increase
45
in lymphocytes (WBCs), neutrophils and platelets. Studies have reported that infections induce
decrease in immune response through the inhibition of CD4+ and CD8+ cells, mainly the
lymphocytes and neutrophils (Yapo et al., 2011). Hence, the results of this study indicate that the
extract has immune stimulating properties, which is essential for HIV positive patients. The
extract may also be a supportive treatment for thrombocytopenic (bleeding) disorders which are
sometimes caused by viral or bacterial infections like Hepatitis C, where the blood does not clot.
There was a significant increase in serum urea and creatinine, which is an indication of
functional damage to the kidneys. Some plants increased urination, a property that has a
significant diuretic effect in rats. Diuretics can be used to treat conditions like hypertension,
kidney, liver diseases and urinary tract infections. Although there was no incidence of mortality
from the toxicity tests implying that the water extracts are relatively safe with low risk of acute
intoxication, histological examinations of the internal organs of rats showed severe toxic effects
after a month’s administration of the extract. However, toxic effects were observed as the
concentration of extracts increased. This means that the extract is toxic at high doses. Hence,
proper formulation of the herbal extract is needed for better treatment results. Data from such
studies is important for any future in vivo and clinical studies of these plants. Chronic toxicity
studies are necessary to support the safe use of these plants. The results support the traditional
use of the plant in boosting immunity.
From the results of acute toxicity tests, it was observed that essential oils showed lower lethal
doses (more toxic) than the water extracts. The weak acute toxicity levels of the water extracts
could be the reason why these plants have long been used for medicine. Many essential oils
being complex mixtures of various compounds could be the reason why they are more toxic.
Many essential oil components are known to be toxic when taken orally (Shaaya et al., 1999).
46
5. Conclusions
Traditional knowledge can lead to useful medicinal plants used to manage and treat various
disease conditions including infections associated with HIV/AIDS. The study recorded a
rich diversity of plant species with potential to treat various ailments including infections
associated with immuno-compromised people living with HIV/AIDS in western Uganda.
Such studies can help stimulate confidence in traditional medicine and enhance appreciation
of herbal medicine among the people and to appreciate the value of the plant resources and
therefore enhance conservation efforts of the plant species. The high consensus values
obtained from the results means the majority of informants agree on the use of plant species
and this reflects the importance of the nutri-medicinal plants to the people. There is urgent
need to document key plant species with medicinal and nutritional values because a number
of them are becoming rare and others are threatened with extinction in their natural habitats.
Ethnopharmacological studies are needed to validate the use of the documented plants
(Papers I, II, III).
This study has shown the presence of phytochemical compounds and antimicrobial activities
of plant extracts. Phenolic compounds, alkaloids and terpenes are highly potent bioactive
compounds and could perhaps be responsible for most activities shown by the studied
plants. It is concluded that the plant extracts possess antimicrobial activity against tested
organisms. The zone of inhibition varied suggesting the varying degree of efficacy and
different constituents of the plants on the target organisms. All the four screened plants were
active against one or two microbial organisms. Plectranthus amboinicus was active against
all the tested organisms and was the only plant that showed activity against Pseudomonas
aeruginosa. The results of the present study seem to be promising and may enhance the
natural products uses, showing the potential of these plants in the treatment of infectious
diseases caused by Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella
pneumoniae, Candida albicans, Cryptococcus neoformans and Pseudomondas aeruginosa.
The results show that Ipomea hilderbrandti expressed only antifungal properties. This is in
support of its use in traditional medicine to treat fungal infections of the skin (Tinea capitis
and Tinea corporis). The study forms a basis for further phytochemical and pharmacological
47
studies to isolate and characterize the bioactive principles necessary for the development of
new antimicrobial drugs. Further studies are needed to carry out bioassay –guided
fractionation of the essential oil in order to isolate the active compounds that can be useful
in management of these fungal infections.
All the ten screened plants showed antioxidant activity in terms of reducing power and
radical scavenging capacity. This study shows that all the plant species contain sufficient
amounts of minerals required daily for maintenance of the immune system. These results
may be useful for the evaluation of dietary information and we conclude that these plants
have the potential to be sources of natural antioxidants and mineral nutrients. The inhibitory
effect of the extracts on free radicals may justify the traditional use of some of the plants as
immunity boosters in the management of opportunistic infections in HIV/AIDS patients.
No mortalities were observed during acute and sub acute toxicity studies. However, results
indicate that some of the extracts caused treatment-related toxicological abnormalities which
increased with dosage. The aqueous extract of P. amboinicus is safe to use as indicated by
the high LD50 value, but should be used with caution at high doses. Therefore, this forms the
basis for chronic toxicity studies in order to formulate a dosage that is safe for human
consumption.
Further research is also needed to isolate the plants’ active compounds in order to
understand their modes of action. The significant activity observed in this study could be
attributed to the interaction of one or more identified metabolites against the test organisms.
48
Challenges and future directions of herbal medicine research: Integrating modern and
traditional medicine
Traditional and modern medicines have much to offer each other despite the differences. For
millennia, people around the world have healed the sick with herbal or animal-based remedies
handed down through generations. Herbal medicine is becoming increasingly used to enhance
general health and well-being, and it is also used alone for specific ailments or with modern
medicine. Traditional and modern practices can be integrated as two branches of medical science
with the ultimate incorporation of elements of both to form a new branch. This incorporation has
been demonstrated to be practicable in many countries particularly in Asian countries such as
China, Japan, Korea and India among others (WHO, 2001). Traditional/ herbal medicine can be
incorporated as an integral part of a country’s formal health care system with each being
separately recognized as legitimate forms of health care within the same framework.
However, one of the most important factors affecting the quality of herbal medicines is the lack
of effective policies on quality assurance in the processing and manufacturing of herbal products
under good manufacturing practices. In addition to this, proof of efficacy and safety for the vast
majority of herbal medicine has not been fully established through an evidence-based approach.
Research and development to isolate therapeutically active ingredients is of critical importance to
the development of medical science. There is need to formulate and develop a national policy on
traditional medicine (TM) to ensure adequate regulatory mechanisms are in place for promoting
and maintaining good practice, safety, efficacy and quality of traditional medicines.
Secrecy of traditional healers is a big problem to research on good remedies. There is need to
coin a suitable patent and royalty system policy conducive to support all parties involved in
novel drug discovery.
49
6. Acknowledgements
All Praises are for the Almighty God, the only to be Praised, whose Blessing and exaltations
flourished my thoughts and enabled me to improve my knowledge up to this level. I offer my
humble and sincerest words of thanks to our Lord Jesus Christ who is forever a torch of
knowledge, wisdom and guidance for humanity. I would like to take this opportunity to thank all
who have contributed in different ways to bring this study to a successful completion.
My heartfelt appreciation goes to the Swedish government through the Swedish International
Development Agency (SIDA) for funding this research. Sincere gratitude to Dr Peter Sundin, Mr
Hossein Aminaey and Pravina Gajjar, coordinators at the International Science Programme,
Sweden. The experiments were conducted at the Royal Institute of Technology, KTH, School of
Chemical Science Engineering, Stockholm, Sweden; at Makerere University and MBN Clinical
Laboratories, Kampala, Uganda. The support and cooperation received from these institutions is
highly appreciated.
My deepest thanks go to my supervisors; Professor Anna-Karin Borg-Karlson, Associate
Professor Maud Kamatenesi Mugisha and Dr Agnes Namutebi for accepting me as their student,
for sharing their vast knowledge with me, for their unending enthusiasm and ability to always
encourage and give me support when needed. I am thankful to the Doctoral Committee
Members; Professor Hannington Oryem-Origa, Associate Professor Esezah Kakudidi and
Associate Professor Robert Byamukama for guidance and encouragement. Very special thanks
go to Professor J. Y. T Mugisha and Associate Professor John Mango who never got tired of
signing my vouchers whenever I needed funds. It was a pleasure to work with you all.
I am grateful to my colleagues at the Department of Chemistry, KTH; Azeem Muhammad (you
were such a blessing, I learnt a lot from you), Tao Zhao, Norihisa Kusumoto, Nancy Cabezas,
Lynda Kirie Eneh, Lina Lundborg, Rushana Murtazina, Raimondas Mozuraitis, Karolina
Axelsson, Anders Molin and Jenny Lindh for interesting discussions, help and nice working
atmosphere. They were such an inspiring group of people from all over the world. I am thankful
to Dr Katrin Putsep, Dr Anders Gustafsson and Dr Abier Sofrata for all the support I received
whenever I would be at Karolinska Institutet, Stockholm, Sweden. My sincere gratitude to
50
Professor Peter Baekstroem for introducing and explaining MPLC technique. To all the technical
group at Makerere University: Nakibuuka Mary, Catherine Twesiime, Atuhaire Collins, Musisi
Lubowa, Korugyendo Emily, Kavuma Peter, Jesca Boonabaana, Ndyanabo Susan and Portase
Rwaburindore; Dr Fred Bwanga, Emmanuel Aboce and Irene Najjingo at MBN clinical
laboratories, Kampala. The moral support from my PhD colleagues is highly acknowledged;
Mubiru Edward, Bulafu Collins, Kakuba Christian, Jamil Ssenku, Addisalem Abathun, Peace
Musiimenta, Christine Kyarimpa, Namukobe Jane, and Madrine Adia Madina. Thank you all.
I cannot ignore my appreciation and deep sense of gratitude from my family, especially my dear
husband Associate Professor Byarugaba Dominic for the love and support; my children for their
patience during the times I was away from home. Taremwa Derrick, Twiine Davis and Asiimwe
Josephine, I love you all. You are wonderful may God bless you. My sincere gratitude to my
dearest parents, Mr and Mrs Apollinari Tirikwendera who remembered me in their prayers. I am
thankful to my sister Helen Musiime who shared my responsibilities during my field work
research. I thank my brothers Alex Mugisha, Emmanuel Mbuga and Agaba Godfrey for
supporting me.
Finally, I want to thank all the local communities of Mbarara, Ibanda, Isingiro and Kiruhura
districts especially Medius Namanya, Jenipher Kabona, Maurice Nturanabo, Nyamwija J,
Kwetegyereza Fred, Paulina Tumuheirwe and all the Local Council Chairpersons for identifying
all the respondents that shared their knowledge with me which has made this project a great
success.
51
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Appendix A: the author’s contribution to the publications
Papers I, II, III: I participated in planning and collecting ethnobotanical data from the study areas
and writing the manuscripts.
Paper IV: I participated in planning of experiments, performed chemical analyses and writing the
manuscript.
Manuscripts V and VI: I participated in planning of experiments, performed chemical analyses
and writing manuscripts.
62
Appendix B: Chemical structures of major compounds in plants with antimicrobial and
antioxidant activities.
Limonene α-phellanderene α-terpinene
O
HO
OH
1,8-cineole carvacrol geraniol
HO
OH
nerol linalool α -caryophyllene
O
HO
β-eudesmene caryophyllene oxide cis, cis- farnesol
63
Appendix C. Preliminary antimicrobial screening of the twenty plant extracts
64
65
66
Appendix D: Antimicrobial screening of the four active plant extracts
Table 1. Zone of inhibition, MIC and MBC of essential oil (dry leaf) of Plectranthus amboinicus on
selected bacterial and fungal species
Test organism Essential oil Ethanol extract
Inhibition
(mm)
Control MIC
(mg/ml)
MBC /MFC
(mg/ml)
Inhibition
(mm)
MIC
(mg/ml)
MBC
(mg/ml)
Klebsiella
pneumoniae
38.5 ± 0.5 31.5 ± 0.5 25 50 Na - -
Staphylococcus
aureus
32.0 ± 0.5 25 ± 0 50 50 Na - -
Streptococcus
pneumoniae
32 ± 0 35 ± 0 50 50 Na - -
Candida albicans 54.4 ± 4.0 15 ± 0 1.56 25 Na - -
Cryptococcus
neoformans
25.0 ± 0 15 ± 0 25 25 Na - -
Inhibition zone diameter (mm) mean ± SEM, n=2; MBC – Minimum Bactericidal activity; MFC – Minimum
fungicidal activity; Na- no activity
Table 2. Zone of inhibition, MIC and MBC of essential oil (fresh leaf) of Plectranthus amboinicus on
selected bacterial and fungal species
Test organism Essential oil Water extract
Inhibition
(mm)
Control MIC
(mg/ml)
MBC /MFC
(mg/ml)
Inhibition
(mm)
MIC
(mg/ml)
MBC
/MFC
(mg/ml)
Klebsiella
pneumoniae
50.0 ± 0 31.5 ± 0.5 50 50 Na - -
Staphylococcus
aureus
Na 25 ± 0 - - 13.0 ± 0 50 50
Streptococcus
pneumoniae
Na 35 ± 0 - - Na - -
Candida albicans 26.0 ± 0 15 ± 0 25 50 Na - -
Cryptococcus
neoformans
17.0 ± 0 15 ± 0 25 50 Na - -
Inhibition zone diameter (mm) mean ± SEM, n=2; MBC – Minimum Bactericidal activity; MFC – Minimum
fungicidal activity; Na- no activity
67
Table 3. Zone of inhibition, MIC and MBC of essential oil of Crassocephalum vitellinum on selected
bacterial and fungal species
Test organism Essential oil & fractions Water extract
Inhibition
(mm)
Control MIC
(mg/ml)
MBC /MFC
(mg/ml)
Inhibition
(mm)
MIC
(mg/ml)
MBC
(mg/ml)
Klebsiella
pneumoniae
10 ± 0 31.5 ± 0.5 50 50 Na - -
Staphylococcus
aureus
14 ± 0 25 ± 0 25 50 15.0 ± 0 25 50
Streptococcus
pneumoniae
25.0 ± 0 35 ± 0 50 50 Na - -
Candida albicans 20 ± 0 15 ± 0 50 50 Na - -
Cryptococcus
neoformans
34.5 ± 2.5 15 ± 0 50 50 Na - -
Cryptococcus
neoformans
Fr 3 (51± 2) 15 ± 0 3.12 50 Na - -
Cryptococcus
neoformans
Fr 4 (50 ± 0) 15 ± 0 25 50 Na - -
Cryptococcus
neoformans
Fr 5 (37 ± 3) 15 ± 0 25 50 Na - -
Table 4. Zone of inhibition, MIC and MBC of essential oil of Erlangea tomentosa on selected bacterial
and fungal species
Test organism Essential oil & fractions Ethanol extract
Inhibition (mm) Control MIC
(mg/ml)
MBC /MFC
(mg/ml)
Inhibition
(mm)
MIC
(mg/ml)
MBC
(mg/ml)
Klebsiella
pneumoniae
15.0 ± 0 31.5 ± 0.5 25 50 Na - -
Klebsiella
pneumoniae
Fr 5 (16 ± 1) 31.5 ± 0.5 50 50 Na - -
Staphylococcus
aureus
16.0 ± 0 25 ± 0 25 50 15 ± 0 50 50
Staphylococcus
aureus
Fr 2 (15 ± 0) 25 ± 0 25 50 Na - -
Staphylococcus
aureus
Fr 3 (15.5 ± 0.5) 25 ± 0 50 50 Na - -
Staphylococcus
aureus
Fr 4 (11 ± 0) 25 ± 0 25 50 Na - -
Candida albicans 30.0 ± 0 15 ± 0 25 50 10 ± 0 50 50
68
Candida albicans Fr 3 (10 ± 0) 15 ± 0 50 50 Na - -
Candida albicans Fr 4 (25 ± 0) 15 ± 0 25 50 Na - -
Candida albicans Fr 5 (18 ± 0) 15 ± 0 25 50 Na - -
Cryptococcus
neoformans
20.0 ± 0 15 ± 0 25 50 Na - -
Cryptococcus
neoformans
Fr 3 (15 ± 0) 15 ± 0 50 50 Na - -
Cryptococcus
neoformans
Fr 4 (34.5 ± 0.5) 15 ± 0 25 50 Na - -
Inhibition zone diameter (mm) mean ± SEM, n=2; MBC – Minimum Bactericidal activity; MFC – Minimum
fungicidal activity; Na- no activity
Table 5. Zone of inhibition, MIC and MBC of essential oil of Ipomea hildebrandtii on selected bacterial
and fungal species
Test organism Essential oil & fractions
Inhibition (mm) Control MIC
(mg/ml)
MBC /MFC
(mg/ml)
Klebsiella
pneumoniae
Na 31.5 ± 0.5 - -
Staphylococcus
aureus
Na 25 ± 0 - -
Streptococcus
pneumoniae
Na 35 ± 0 - -
Candida albicans 32.0 ± 0 15 ± 0 12.5 25
Candida albicans Fr 2 (28 ± 0) 15 ± 0 25 50
Cryptococcus
neoformans
10.0 ± 0 15 ± 0 50 50
Inhibition zone diameter (mm) mean ± SEM, n=2; MBC – Minimum Bactericidal activity;
MFC – Minimum fungicidal activity; Na- no activity
69
Appendix E: toxicity signs observed in mice during acute toxicity study of the essential oils of
the plant leaves (treatment-related changes)
Plant species name Dose (mg/kg) Observed signs of toxicity
Plectranthus amboinicus 5000 drowsiness, ataxia, paralysis, numbness, irritation and
hyperurination
6500 Hypoactivity, drowsiness, ataxia, paralysis, numbness,
irritation and hyperurination
Pseudarthria hookeri 4500 Paralysis of hind limbs
5500 Circular movements
6500 Died in 24 hours
Plunchea ovalis 5000 Hyperurination, hypoactivity
6500 Paralysis
7500 Paralysis, died in 4 hours
8500 Convulsions, lameness, paralysis, died in 10 minutes
Symphytum officinale 2000 Irritation
3500 Paralysis, died in 2 hrs
4000 Died in 2 hrs
4500 Severe paralysis, died in 2 hrs
5000 Weak with facial swollenness, died in 1 hr
Tarenna pavettoides 5000 Circling movements
5500 Circling movements, died in 2 hrs
7500 Paralysis, increased heartbeat, irritation, died in 1 hr
70
Appendix F. Chromatograms and mass spectra of essential oils of some active plant species
RT: 0.00 - 75.01
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
48.28
9.62
12.1030.82
12.99 24.8848.88
32.74
29.2022.19
51.07
5.01 47.4251.5646.89
36.26 42.97
9.074.38 18.01 39.3452.45 58.38 58.7813.46 69.24 73.79
NL:1.65E8
TIC MS AP-Ess-oil
(replib) Phenol, 2-methyl-5-(1-methylethyl)-
30 40 50 60 70 80 90 100 110 120 130 140 150 1600
50
100
3139 51 65
7782 87
91107 115
135
150HO
Chromatogram of the essential oil of Plectranthus amboinicus showing carvacrol (14.3%) as the
abundant compound
71
RT: 0.00 - 75.01
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
40.53
9.0810.21
42.41
30.69
24.77
29.09 33.21
26.287.5546.56 52.994.98
45.3359.8636.05
48.7222.06
19.16 49.24 67.514.35 12.28 59.46
12.86
66.11 70.3715.70
NL:8.62E7
TIC MS AC-Ess-oil
(replib) Caryophyllene oxide
40 60 80 100 120 140 160 180 200 2200
50
10043
55
6979 93
109
121
138149 161 177
205 220
O
Chromatogram and mass spectra of the essential oil of Crassocephalum vitellinum showing
caryophyllene oxide (9.5%) as the abundant compound